Compounds and methods for the diagnosis and treatment of amyloid associated diseases

ABSTRACT

The invention is in general directed to compounds and methods for diagnosing, preventing or alleviating the symptoms of amyloid-associated diseases, for example, neuronal diseases, such as, for example, Alzheimer&#39;s disease, methods of screening for compounds useful in preventing or alleviating the symptoms of amyloid-associated diseases, methods of diagnostic imaging of A-beta fibrils, and compounds and methods useful for studying normal or disease-associated cellular mechanisms relating to amyloid proteins.

FIELD OF THE INVENTION

The invention is in general directed to compounds and methods fordiagnosing, preventing or alleviating the symptoms of amyloid-associateddiseases, for example, neuronal diseases, such as, for example,Alzheimer's disease, methods of screening for compounds useful inpreventing or alleviating the symptoms of amyloid-associated diseases,methods of diagnostic imaging of A-beta fibrils, and compounds andmethods useful for studying normal or disease-associated cellularmechanisms relating to amyloid proteins.

PRIORITY

Priority is claimed to U.S. Provisional Application Ser. No. 60,699,726,filed Jul. 15, 2005, and entitled New Approach for Therapeutics forAlzheimer's Disease; and U.S. Provisional Application Ser. No.60,750,422, filed Dec. 13, 2005, and entitled Compounds and Methods forthe Diagnosis and Treatment of Alzheimer's Disease, which are bothreferred to and incorporated herein by reference in their entirety.

BACKGROUND

Amyloid fibrils formed from misfolded proteins or peptides are ahallmark of many neuronal diseases, such as, for example, Alzheimer'sdisease. (Soto, C. Nature Rev. Neurosci. 2003, 4: 49; Agorogiannis, E.I., et al., Neuro path. Appl. Neurobiol. 2004, 30:215; Kelly, J. W.Structure 1997, 5:595.) Amyloid fibrils have also been associated withother, non-neuronal diseases and conditions, such as, for example, thoselisted in Table 1.

Amyloid fibrils and plaques are rich in beta sheet structure. A-beta isa peptide found in amyloid fibrils and plaques. Researchers haveassociated the development of Alzheimer's disease (AD), with theinteraction of A-beta peptides, oligomers, and fibrils with cellularcomponents in the brain. (Dawbarn, D., and Allen, S. J. Neurobiology ofAlzheimer's disease, second ed., Oxford University Press, Oxford, 2001;Pereira, C., et al., J. Mol. Neurosci. 2004, 23: 97.) The interactionbetween cellular proteins, such as, for example, catalase, ABAD (betaamyloid-binding alcohol dehydrogenase) and RAGE (receptor for advancedglycation end products) and aggregated A-beta-amyloid fibrils (A-betafibrils), for example, have been reported for their potentialcontribution to A-beta-induced neurotoxicity in the pathogenesis of AD.(Milton, N. G. N. Biochem. J. 1999, 344:293-296; Milton, N. G. N., etal. Neuroreport 2001, 121: 2561; Yan, S. D., et al. Nature 1997, 389:689; Yan, S. D., et al. J. Biol. Chem. 1999, 274: 2145; Lustbader, J.W., et al. Science 2004, 304: 448; Yan, S. D., et al. Nature 1996, 382:685; Yan, S. D, et al., Am. J. Pathol. 1999, 155: 1403; Yan, S. D., etal., Biochim. Biophys. Acta 2000, 1502: 145; K. Takuma, J. Yao, J.Huang, H. Xu, X. Chen, J. Luddy, A.-C. Trillat, D. M. Stern, O. Arancio,S. S. Yan, FASEB J. 2005, 19(6), 597-598; Takuma, K., et al., FASEB J.2005, 19(6): 597-598)

Several classes of small molecule therapeutics are used clinically totreat the symptoms of AD, such as, for example, inhibitors ofcholinesterase. (Francis, P. T., et al, Trends Pharm. Sci. 2005, 26:104; Conway, K. A., et al., Curr. Pharm. Design 2003, 9: 427.) Currentstrategies to modify directly the pathology of AD using syntheticmolecules are focused mainly on slowing down the production of A-betapeptide or preventing the growth of A-beta fibrils. (C. Schmuck, et al.,ChemBioChem 2005, 6: 1; C. N. Johnson, et al., Drug Dis. Today 2004, 1:13; M. S. Parihar and T. Hemnani, J. Clin. Neurosci. 2004, 11: 456; V.M.-Y. Lee, Neurobio. Aging 2002, 23: 1039; B. Bohrmann, et al., J. Biol.Chem. 1999, 274: 15990; F. G. De Felice, et al., FASEB J. 2004, 18:1366;M. A. Findeis, Biochim. Biophys. Acta 2000, 1502:76; J. E. Gestwicki, etal., Science 2004, 306: 865).

Other strategies focus on disrupting the fibrils so that theydisassemble into their A-beta peptide components. These approaches mayincrease the amount of A-beta peptide, A-beta-dimers, or small A-betaoligomers in neurons, which may have a toxic affect.

Thioflavin T (ThT) a fluorescent molecule (FIG. 1 b)—is used extensivelyfor the characterization of A-beta fibrils (LeVine III, H. Meth. Enzym.1999, 309: 274.) and for the detection of aggregation of A-beta insolution. (Blanchard, B. J., et al., Proc. Nat. Acad. Sci. USA 2004,101: 14326; Ono, K., et al., J. Neurochem. 2002, 81: 434.) Severalgroups have studied the interaction of ThT with A-beta fibrils byfluorescence and showed that ThT binds uniformly to the bulk of A-betafibrils with high affinity (K_(d)'s ranging from high nM to low μM).(LeVine III, H. Protein Sci. 1993, 2: 404; LeVine III, H. Amyloid 1995,2: 1; LeVine III, H. Arch. Biochem. Biophys. 1997, 342: 306; Lockhart,A., et al., J. Biol. Chem. 2005, 280: 7677; Krebs, M. R. H., et al., J.Struct. Biol. 2005, 149: 30). Thioflavin derivatives have been reportedto be used in the diagnosis of Alzheimer's and in in vivo imaging, andCongo Red (CR) is used by researchers to stain amyloid plaques found inpatients with Alzheimer's disease. (Klunk, W., et al., U.S. PatentApplication Publication No. 20050043377 (2005); C. A. Mathis, et al.,Current Pharm. Design, 2004, 10:1469-92; and Hintersteiner, M., et al.,Nature Biotechnology, 2005, 23:577-83.)

Molecular coatings on metallic and polymeric surfaces are usedfrequently to attenuate interactions of proteins with artificialmaterials for biological studies and biotechnology applications.(Chapman, R. G., et al., J. Am. Chem. Soc. 2000, 122: 8303; Mrksich, M.,et al., Proc. Nat. Acad. Sci. USA 1996, 93:10775; Chiu, D. T., et al.,Proc. Nat. Acad. Sci. USA 2000, 97: 2408; Chen, X., et al., Langmuir2002, 18: 7009; Siegers, C., et al., Chem. Eur. J. 2004, 10: 2831.)

There is a need for novel methods and compounds for diagnosing andtreating amyloid-associated diseases, for example, neuronal diseases andconditions, with a smaller incidence of toxicity.

SUMMARY

Provided herein are compounds and methods for preventing or alleviatingthe symptoms of amyloid-associated diseases, for example, but notlimited to, neuronal diseases and conditions associated with amyloidfibril or plaque formation. It has been found that providing a bindingmolecule that coats the surface of an A-beta fibril may allow thefibrils to resist the interaction of cellular proteins with thesefibrils, resulting in a new strategy to intervene in AD-relatedpathology. Provided herein are compounds that inhibit the bindinginteraction between A-beta fibrils and cellular proteins. In otherembodiments are provided methods of screening for compounds useful inpreventing or alleviating the symptoms of neuronal diseases, and methodsof diagnostic imaging of A-beta fibrils.

In a first embodiment of the present invention is provided a compound offormula I:

-   -   wherein R₁-R₈ are selected from the group consisting of        hydrogen, deuterium, tritium, fluoride, chloride, bromide,        iodide, hydroxide, amino, methylamino, dimethylamino,        trimethylammonium, methyl, ethyl, methoxy, ethoxy, fluoromethyl,        difluoromethyl, trifluoromethyl, wherein at least one of R5-R8        and one of R₁-R₄ is H; and    -   P is selected from the group consisting of

-   -   wherein    -   m is an integer between 1 and 20;    -   n is 0, 1, or 2;    -   q is an integer between 1 and 20;    -   R₉-R₁₆ are selected from the group consisting of hydrogen,        deuterium, tritium, fluoride, chloride, bromide, iodide,        hydroxide, amino, methylamino, dimethylamino, trimethylammonium,        methyl, ethyl, methoxy, ethoxy, fluoromethyl, difluoromethyl,        trifluoromethyl, wherein at least one of R₉-R₁₂ and one of        R₁₃-R₁₆ is H; and    -   X is hydrogen, methyl, or ethyl.        In further embodiments, P, for example, is

In exemplary embodiments of the present invention, for example, m is aninteger between 3 and 16, 3 and 12, 5 and 10, 6 and 10, 3 and 7, 4 and7, or 3 and 8. By “between” in the context of the present invention, ismeant to include both the first and second numbers. Thus, “between 4 and6” is meant to include 4, 5, and 6. In exemplary embodiments, m is 4,for example, or m is 6, for example. In other exemplary embodiments,R₁-R₁₆ are H. In exemplary embodiments of the present invention, thecompound is BTA-EG₄, BTA-EG₆, BTA-Aza-Crown₅, or BTA-EG₆-BTA. By“compound of the present invention” is meant a compound of formula I,including, for example, each of the embodiments of such compounds.

In other examples of the present invention, P is

and n is 1.

The present invention further provides pharmaceutical compositionscomprising a pharmaceutically acceptable excipient and any of thecompounds of the present invention.

Also provided in the present invention is a method of preventing oralleviating the symptoms of an amyloid associated disease comprisingcontacting A-beta fibrils with a compound of the present invention. Inexemplary embodiments, the disease is a neuronal disease. In furtherexemplary embodiments, the neuronal disease is selected from the groupconsisting of Alzheimer's disease, Parkinson's disease, Huntington'sdisease Down's Syndrome, and spongiform encephalopathy. For example, theneuronal disease may be, but is not limited to, Alzheimer's disease. Or,for example, the neuronal disease may be, but is not limited to,Parkinson's disease.

Also provided in the present invention is a method of preventing oralleviating the symptoms of an amyloid associated disease comprisingcontacting A-beta fibrils with a sufficient amount of a first bindingmolecule to decrease the interactions of the A-beta fibrils with asecond binding molecule. In certain embodiments, the disease is aneuronal disease. In certain embodiments, a plurality of the firstbinding molecules forms an ordered layer on top of the fibrils. Forexample, the first binding molecule may coat a portion of the surface ofthe fibrils. The first binding molecule may, for example, coat more than50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of the surface of thefibrils. The second binding molecule may be, for example a cellularcomponent in the brain. The second binding molecule may be, for example,a cellular protein. The second binding molecule may be, for example,selected from the group consisting of catalase, ABAD, and RAGE. Incertain embodiments of the present invention, the binding of the secondbinding molecule to the fibrils is associated with the symptoms of anamyloid associated disease, such as, for example, those listed inTable 1. In other embodiments of the present invention, the binding ofthe second binding molecule to the fibrils is associated with thesymptoms of a neuronal disease, such as, for example, Alzheimer'sdisease, Parkinson's disease, Huntington's disease Down's Syndrome, orspongiform encephalopathy. In exemplary embodiments, the neuronaldisease is Alzheimer's disease. In other exemplary embodiments, theneuronal disease is Parkinson's disease.

In some aspects of the present invention, the first binding moleculebinds to the fibrils using hydrophobic and electrostatic interactions.In other aspects, the first binding molecule binds to the fibrils usingnon-covalent interactions with the fibrils. In certain aspects of theinvention, the first binding molecule is selected from the groupconsisting of Congo Red, a Congo Red derivative, Thioflavin T, and aThioflavin T derivative. In other aspects of the invention, the firstbinding molecule is a compound of the present invention. In furtherembodiments of the invention, the method comprises administering atherapeutically effective amount of the first binding molecule to anindividual. By “individual” is meant, for example, any animal, forexample, any mammal, such as, for example, a bovine, rodent, primate,horse, canine, feline, or human. In exemplary embodiments, theindividual is human.

Also provided in the present invention is a method of screening for acompound that blocks the binding of an A-beta fibril binding molecule toA-beta fibrils, comprising

-   -   Depositing A-beta fibrils onto a test surface.    -   Adding a test compound to the A-beta fibrils and incubating the        deposited fibrils with the test compound;    -   Adding an A-beta fibril binding molecule to the test surface;    -   Determining the amount of the binding molecule that binds the        fibrils; and    -   Determining if the test compound decreases the binding of the        binding molecule to the fibrils.

In other embodiments of the present invention is provided a method ofscreening for a compound that blocks the binding of an A-beta-fibrilbinding molecule to A-beta fibrils, comprising

-   -   Incubating A-beta fibrils with a test compound;    -   Depositing the test-compound-incubated A-beta fibrils onto a        test surface;    -   Adding an A-beta fibril binding molecule to the test surface;    -   Determining the amount of the binding molecule that binds the        fibrils; and    -   Determining if the test compound decreases the binding of the        binding molecule to the fibrils.        The compounds of the present invention may also be used for        diagnostic imaging of A-beta fibrils. The methods of the present        invention also provide methods for screening for compounds        useful for diagnostic imaging of A-beta fibrils, comprising, for        example,    -   Depositing A-beta fibrils onto a test surface;    -   Adding a test compound to the A-beta fibrils and incubating the        deposited fibrils with the test compound;    -   Adding an A-beta fibril binding molecule to the test surface;    -   Determining the amount of the binding molecule that binds the        fibrils; and    -   Determining if the test compound decreases the binding of the        binding molecule to the fibrils.

In other embodiments of the present invention is provided a method ofscreening for a compound used for diagnostic imaging of A-beta fibrils,comprising

-   -   Incubating A-beta fibrils with a test compound;    -   Depositing the test-compound-incubated A-beta fibrils onto a        test surface;    -   Adding an A-beta fibril binding molecule to the test surface;    -   Determining the amount of the binding molecule that binds the        fibrils; and    -   Determining if the test compound decreases the binding of the        binding molecule to the fibrils.

The test surface may be, for example, a test well. The test well, maybe, for example, present in a microtiter plate. The A-beta fibrilbinding molecule, in exemplary embodiments, may be, for example,selected from the group consisting of an antibody, RAGE, ABAD, andcatalase. For example, the antibody may be an anti-A-beta antibody.Determining the amount of A-beta fibril binding molecule that binds thefibrils may be conducted, for example, using an ELISA assay. In certainaspects of the method, prior to adding the test compound to the well,the test compound is pre-incubated with A-beta-fibrils. Those ofordinary skill in the art will understand that various concentrations ofthe test compound may be tested in the screening, and that determiningwhether the test compounds decreases binding of a binding molecule tothe fibrils may be conducted by comparing the amount of binding of thebinding molecule to the fibrils in the presence and in the absence ofthe test compound. This may also include, for example, comparing theamount of binding of the binding molecule to the fibrils in the presenceof at least two different concentrations of the test compound. Incertain exemplary screening embodiments, the A-beta fibril bindingmolecule is an isolated brain cellular component, for example, acomponent selected from the group consisting of catalase, ABAD, andRAGE. The test compound may be, for example, a compound of the presentinvention.

In yet other embodiments of the present invention are provided methodsfor diagnosing an amyloid associated disease in an individual,comprising administering an A-beta fibril-binding compound to anindividual and detecting the binding of the compound to amyloid depositsin the individual, wherein the compound is selected from the groupconsisting of a compound of any of claims 1-6, Congo Red, Fluoroscein,Acridine Orange, Diamino Acridine, Crystal Violet, Thioflavin T,Oxytetracycline, Tetracycline, Chlortetracycline, Tannic Acid,Rosmarinic Acid, (+)-Catechin, (−)-Nicotine, Morin, Serotonin, Dopamine,Curcumin, (R)-Ibuprofen, (S)-Naproxen, Rifampin, D-(+)-Trehalose,D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof. In exemplary embodiments, the amyloid associated disease is aneuronal disease. In further exemplary embodiments, the compound is acompound of the present invention.

In further embodiments of the present invention are provided methods foridentifying a change in the progress of an amyloid associated disease inan individual, comprising

-   -   administering an A-beta fibril-binding compound to an individual        and conducting a first detecting procedure to detect the binding        of the compound to amyloid deposits in the individual on a first        date;    -   administering an A-beta fibril binding compound to the        individual and conducting a second detecting procedure to detect        the binding of the compound to amyloid deposits in the        individual on a second date; and    -   comparing the amount, quantity, or other characteristics of the        amyloid deposits detected in step b with the amyloid deposits        detected in step a, wherein the A-beta fibril binding compound        is selected from the group consisting of a compound of the        present invention, Congo Red, Fluoroscein, Acridine Orange,        Diamino Acridine, Crystal Violet, Thioflavin T, Oxytetracycline,        Tetracycline, Chlortetracycline, Tannic Acid, Rosmarinic Acid,        (+)-Catechin, (−)-Nicotine, Morin, Serotonin, Dopamine,        Curcumin, (R)-Ibuprofen, (S)-Naproxen, Rifampin,        D-(+)-Trehalose, D-nicotine, pegylated derivatives of        1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and        analogs thereof. In exemplary embodiments, the disease is a        neuronal disease. In certain embodiments, the compound is        selected from the group consisting of a compound of the present        invention, Congo Red, Fluoroscein, Acridine Orange, Diamino        Acridine, Crystal Violet, Thioflavin T, Oxytetracycline,        Tetracycline, Chlortetracycline, Tannic Acid, Rosmarinic Acid,        (+)-Catechin, (−)-Nicotine, Morin, Serotonin, Dopamine,        Curcumin, (R)-Ibuprofen, (S)-Naproxen, Rifampin,        D-(+)-Trehalose, D-nicotine, and pegylated derivatives of        1-(p-aminophenyl)-6-methylbenzothiazole. In exemplary        embodiments, the compound is a compound of the present        invention.

In yet further embodiments of the present invention are provided methodsfor detecting amyloid deposits in an individual, comprising

-   -   administering a pharmaceutical composition comprising a        pharmaceutically acceptable carrier and a compound selected from        the group consisting of a compound of the present invention,        Congo Red, Fluoroscein, Acridine Orange, Diamino Acridine,        Crystal Violet, Thioflavin T, Oxytetracycline, Tetracycline,        Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,        (−)-Nicotine, Morin, Serotonin, Dopamine, Curcumin,        (R)-Ibuprofen, (S)-Naproxen, Rifampin, D-(+)-Trehalose,        D-nicotine, pegylated derivatives of        1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and        analogs thereof; and    -   detecting the binding of the compound to an amyloid deposit in        the individual. In exemplary embodiments, the pharmaceutical        composition comprises a compound of the present invention. In        certain embodiments, the amyloid deposit is present in the brain        of the individual.

For detecting the presence of amyloid deposits, for example, the A-betafibril-binding compound may be, for example, radiolabeled. Detection maybe conducted by a method, for example, selected from the groupconsisting of gamma imaging, magnetic resonance imaging, or magneticresonance spectroscopy. The detection may be, for example, single photonemission computed tomography or positron emission tomography.

In other embodiments of the present invention are provided methods forpreventing or alleviating the symptoms of an amyloid associated diseasecomprising contacting A-beta fibrils with a sufficient amount of apharmaceutical composition comprising a pharmaceutically acceptablecarrier and an A-beta fibril binding compound selected from the groupconsisting of a compound of the present invention, Congo Red,Fluoroscein, Acridine Orange, Diamino Acridine, Crystal Violet,Thioflavin T, Oxytetracycline, Tetracycline, Chlortetracycline, TannicAcid, Rosmarinic Acid, (+)-Catechin, (−)-Nicotine, Morin, Serotonin,Dopamine, Curcumin, (R)-Ibuprofen, (S)-Naproxen, Rifampin,D-(+)-Trehalose, D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof, to decrease the interactions of the A-beta fibrils with asecond binding molecule. In exemplary embodiments, the disease is aneuronal disease. In exemplary embodiments, the pharmaceuticalcomposition comprises a compound of the present invention.

In other embodiments of the present invention are provided methods forpreventing or alleviating the symptoms of an amyloid associated diseasein an individual comprising administering to the individual atherapeutically effective dose of a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and an A-beta fibrilbinding protein selected from the group consisting of a compound of thepresent invention, Congo Red, Fluoroscein, Acridine Orange, DiaminoAcridine, Crystal Violet, Thioflavin T, Oxytetracycline, Tetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,(−)-Nicotine, Morin, Serotonin, Dopamine, Curcumin, (R)-Ibuprofen,(S)-Naproxen, Rifampin, D-(+)-Trehalose, D-nicotine, pegylatedderivatives of 1-(p-aminophenyl)-6-methylbenzothiazole, and derivativesand analogs thereof, in a pharmaceutically acceptable carrier. Inexemplary embodiments, the disease is a neuronal disease. In exemplaryembodiments, the pharmaceutical composition comprises a compound of thepresent invention. The neuronal disease may be, for example, Alzheimer'sdisease; the neuronal disease may be, for example, Parkinson's disease.

In other exemplary embodiments of the present invention is provided acomposition comprising a compound bound to one or more A-beta fibrils,wherein the compound is selected from the group consisting of a compoundof the present invention, Fluoroscein, Diamino Acridine, Crystal Violet,Oxytetracycline, Tetracycline, Chlortetracycline, Tannic Acid,Rosmarinic Acid, (+)-Catechin, (−)-Nicotine, Morin, Serotonin, Dopamine,Curcumin, (R)-Ibuprofen, (S)-Naproxen, Rifampin, D-(+)-Trehalose,D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof. In exemplary aspects, the compound is a compound of the presentinvention.

Also provided in the present invention are research reagents comprisinga compound of the present invention. The research reagent may be, forexample, formulated to detect amyloid proteins in vivo. The researchreagent may be, for example, formulated to detect amyloid proteins incells or tissue, wherein the cells or tissue have been isolated from aliving organism. Also provided in the present invention is a kitcomprising a research reagent of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Illustration of the inhibition of binding of Aβ-binding proteinsto Aβ fibrils using small molecules. a) In this cartoon, the smallmolecules compete with the Aβ-binding proteins for binding to the Aβfibril (see Puchtler, H., et al., J. Histochem. Cytochem. 1962, 10: 355;LeVine III., H. Meth. Enzym. 1999, 309: 274); b) chemical structures ofThioflavin T (ThT) and two derivatives of2-(4-aminophenyl)-benzothiazoles (BTA-EG₄ and BTA-EG₆).

FIG. 2. Inhibition (Inhib.) of IgG-A-beta interactions with ThT. a)A-beta fibrils incubated with solutions of ThT and exposed to ananti-A-beta IgG (clone 6E10). b) Same assay as in (a) but using ananti-A-beta IgG raised against a different binding epitope of A-betapeptide (clone AMY-33). c) Same assay as in (a) except the inhibition isplotted against the concentration of 1-naphthol-4-sulfonate (NS) insteadof ThT.

FIG. 3. Inhibition (Inhib.) of IgG-A-beta interactions as a function ofincreasing concentrations (conc.) of ThT. ThT and the fibrils wereincubated together prior to depositing the ThT-coated fibrils into96-well plates and exposure to an anti-A-beta IgG (clone 6E10, derivedfrom A-beta residues 3-8 as antigens).

FIG. 4. Compounds used, and results from, a high throughput assay forthe inhibition of IgG-A-beta interactions. Compounds are indicated bynumber and name. IC₅₀s listed give an estimate of the concentrationrange of the compounds required for binding to fibrils, as well as theaffinity of molecules to A-beta fibrils relative to each other. Maxinhibition (Max Ink) gives an estimate of the percentage of the surfaceof A-beta fibrils coated by the small molecules. Not shown in theFigure, but also demonstrating good efficacy in the assay areD-nicotine, oligoethylene derivatives and pegylated derivatives of2-(p-aminophenyl)-6-methylbenzothiazole. FIG. 5. Inhibition (Inhib.) ofIgG-Aβ fibril interactions using small molecules. Aβ fibrils wereincubated with a solution of a monoclonal anti-Aβ IgG (clone 6E10) priorto exposure to solutions of a) BTA-EG₄; and b) BTA-EG₆. Note: the x-axiswas plotted in a logarithmic scale for better visualization of the data.

FIG. 6. Inhibition (Inhib.) of catalase-Aβ fibril interactions usingsmall molecules. a) Binding of Aβ fibrils to catalase. Inhibition ofcatalase-Aβ fibril interactions with solutions of b) ThT; c) BTA-EG₄;and d) BTA-EG₆. Note: the x-axis was plotted in a logarithmic scale forbetter visualization of the data.

FIG. 7. Inhibition (Inhib.) of ABAD-Aβ fibril interactions using smallmolecules. a) Binding of Aβ-fibrils to ABAD. Inhibition of ABAD-Aβfibril interactions with solutions of b) ThT; c) BTA-EG₄; and d)BTA-EG₆. Note: the x-axis was plotted in a logarithmic scale for bettervisualization of the data.

FIG. 8. Inhibition of ABAD-A-β fibril interaction with BTA Aza-crown-5(A) and BTA-EG₆-BTA.

FIG. 9: Inhibition of alpha-synuclein fibril interaction with CongoRed.

DETAILED DESCRIPTION

Provided herein are compounds and methods for diagnosing, preventing oralleviating the symptoms of amyloid-associated diseases, for example,diseases listed in Table 1. In exemplary embodiments are providedcompounds and methods for diagnosing, preventing, or alleviating thesymptoms of neuronal diseases, such as, for example, Alzheimer'sdisease, methods of screening for compounds useful in preventing oralleviating the symptoms of amyloid-associated diseases, methods ofdiagnostic imaging of A-beta fibrils, and compounds and methods usefulfor studying normal or disease-associated cellular mechanisms relatingto amyloid proteins.

Diseases

By “amyloid associated diseases” is meant any disease or condition thatis associated with the increased or decreased presence of amyloidproteins, such as the presence of amyloid plaques. The methods of thepresent invention may be used to diagnose or to detect a propensity foran amyloid-associated disease where no plaques are detected, such as,for example, by detecting amyloid protein as a biomarker. For example,the presence of amylin may be detected using the methods of the presentinvention, and this may be associated, for example, with a likelihood ofdeveloping type-two diabetes. Examples of amyloid associated diseasesmay be found in, but are not limited to, for example, Table 1.

Neuronal diseases that may be diagnosed, treated, prevented or exhibitan alleviation of symptoms according to the present invention includeany neuronal disease or condition, including, for example,neurodegenerative diseases, in which A-beta peptides, oligomers,fibrils, or plaques are implicated, for example, but not limited to,Alzheimer's disease, Parkinson's disease, Huntington's disease, Down'sSyndrome, and spongiform encephalopathies such as, for example, BovineSpongiform Encephalopathy (mad cow disease), Kuru, Creutzfeldt-Jakobdisease, and Fatal Familial Insomnia.

TABLE 1 Amyloid Disease Reference Amylin Type 2 Diabetes Goldsbury, C.S., et al., J Struc. Biol. 1997, 119(1), 17-27; Goldsbury, C., et al.,J. Struc. Biol. 2000, 130(2-3), 352-362; Jimenez, J. L., et al., Proc.Natl. Acad. Sci. 2002, 99(14), 9196-9201. Insulin Type 2 Diabetes Sipe,J. D., Ann. Rev. Biochem. 1992, 61, 947-975 Immunoglobin AL amyloidosisBellotti, V., et al., J. Struc. light chains (liver) Biol. 2000,130(2-3), 280-289; Sipe, J. D., Ann. Rev. Biochem. 1992, 61, 947-975Amyloid A Reactive systemic Sipe, J. D., Ann. Rev. Biochem.(Lipoprotein) amyloidosis 1992, 61, 947-975 Transthyretin senilesystemic Sipe, J. D., Ann. Rev. Biochem. amyloidosis (SSA), 1992, 61,947-975; Brito, familial amyloid R. M. M. et al., Curr. Med.polyneuropathy (FAP) Chem. Immun. Endoc. Metab. and familial amyloidAgents 2003, 3(4), 349-360; cardiomyopathy (FAC) Damas, A. M. andSaraiva, M. J., J. Struc. Biol. 2000, 130(2-3), 290-299; Buxbaum, J. N.,Curr. Opin. Rheumatol. 2003, 16(1), 67-75. β2 Dialysis, renal Buxbaum,J. N., Curr. Opin. microglobulin failure Rheumatol. 2003, 16(1), 67-75.Apolipoprotein Coronary heart Buxbaum, J. N., Curr. Opin. A1 disease,Rheumatol. 2003, 16(1), atherosclerosis 67-75. PrPSc (Prion disease,Wille, H., et al., J. Struc, Biol. sheep) 2000, 130(2-3), 323-338α-synuclein Parkinson's, El-Agnaf, O. M. A. and Irvine, Alzheimer's G.B., J. Struc. Biol. 2000, 130(2-3), 300-309 Cystatin C Cerebralhemorrhage Sipe, J. D., Ann. Rev. Biochem. 1992, 61, 947-975

Methods

Compounds that may be used in the methods of the present inventioninclude compounds found to bind to A-beta fibrils that prevent othercellular components from binding to the fibrils. Compounds that may beused in the methods of the present invention may, for example, have oneor more of the following characteristics: low molecular weight, knownand favorable pharmacokinetic properties, and known permeability acrossthe blood-brain barrier.

Compounds that may be used in the methods of the present invention mayinclude, for example, compounds of the present invention, including, forexample, those listed in the embodiments presented herein.

Compounds that may be used in the methods of the present invention alsoinclude, for example, Congo Red, Congo Red derivatives, ThT, and ThTderivatives. Compounds that may be used in the methods of the presentinvention may include, for example, compounds listed in C. A. Mathis, etal., Current Pharm. Design, 2004, 10:1469-92; and Hintersteiner, M., etal., Nature Biotechnology, 2005, 23:577-83. Compounds that may be usedin the methods of the present invention may be radiolabeled, forexample, for diagnostic imaging, such as that performed using singlephoton emission computed tomography (SPECT) or positron emissiontomography (PET). In illustrative embodiments, the compounds have theability to cross the blood brain barrier. (Di, L., et al., Curr. Opin.Chem. Bio. 2003, 7(3), 402-408; Abraham, M. H., Eur. J. Med. Chem. 2004,39(3), 235-240; Mathis, C. A., et al., Current Pharm. Design, 2004,10:1469-92) Compounds that may be used in the methods of the presentinvention include, for example, those showing inhibitory activity aspresented in FIG. 4, such as, for example, compounds selected from thegroup consisting of Congo Red, Fluoroscein, Acridine Orange, DiaminoAcridine, Crystal Violet, Thioflavin T, Oxytetracycline, Tetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,(−)-Nicotine, Morin, Serotonin, Dopamine, Curcumin, (R)-Ibuprofen,(S)-Naproxen, Rifampin, D-(+)-Trehalose, D-nicotine, pegylatedderivatives of 1-(p-aminophenyl)-6-methylbenzothiazole, and derivativesand analogs thereof. Compounds that may be used in the present inventioninclude, for example, those compounds showing a greater inhibitoryactivity as shown in FIG. 4, such as, for example, compounds selectedfrom the group consisting of Fluoroscein, Acridine Orange, DiaminoAcridine, Crystal Violet, Thioflavin T, Oxytetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,(−)-Nicotine, Morin, Serotonin, Dopamine, Curcumin, (R)-Ibuprofen,Rifampin, D-(+)-Trehalose, D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof. Compounds that may be used in the present invention include,for example, those compounds showing a greater inhibitory activity asshown in FIG. 4, such as, for example, Fluoroscein, Acridine Orange,Diamino Acridine, Crystal Violet, Thioflavin T, Oxytetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,(−)-Nicotine, Serotonin, Dopamine, Curcumin, Rifampin, D-(+)-Trehalose,D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof. Compounds that may be used in the present invention include,for example, those compounds showing a greater inhibitory activity asshown in FIG. 4, such as, for example, Fluoroscein, Acridine Orange,Diamino Acridine, Crystal Violet, Thioflavin T, Oxytetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,(−)-Nicotine, Dopamine, D-(+)-Trehalose, and derivatives and analogsthereof. Compounds that may be used in the present invention include,for example, those compounds showing a greater inhibitory activity asshown in FIG. 4, such as, for example, Fluoroscein, Acridine Orange,Diamino Acridine, Crystal Violet, Oxytetracycline, Chlortetracycline,Tannic Acid, (+)-Catechin, (−)-Nicotine, Dopamine, and derivatives andanalogs thereof. Compounds that may be used in the present inventioninclude, for example, those compounds showing a greater inhibitoryactivity as shown in FIG. 4, such as, for example, Fluoroscein, AcridineOrange, Crystal Violet, Oxytetracycline, (+)-Catechin, (−)-Nicotine,Dopamine, and derivatives and analogs thereof.

In other examples of the present invention, compounds that may be usedto prevent or alleviate the symptoms of a neuronal disease such as, forexample, Alzheimer's disease include, for example, compounds selectedfrom the group consisting of Congo Red, Fluoroscein, Acridine Orange,Diamino Acridine, Crystal Violet, Thioflavin T, Oxytetracycline,Tetracycline, Chlortetracycline, Tannic Acid, Rosmarinic Acid,(+)-Catechin, (−)-Nicotine, Morin, Serotonin, Dopamine, Curcumin,(R)-Ibuprofen, (S)-Naproxen, Rifampin, D-(+)-Trehalose, D-nicotine,pegylated derivatives of 1-(p-aminophenyl)-6-methylbenzothiazole, andderivatives and analogs thereof. Compounds that may be used in thepresent invention include, for example, those compounds showing agreater inhibitory activity as shown in FIG. 4, such as, for example,compounds selected from the group consisting of Fluoroscein, AcridineOrange, Diamino Acridine, Crystal Violet, Thioflavin T, Oxytetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,(−)-Nicotine, Morin, Serotonin, Dopamine, Curcumin, (R)-Ibuprofen,Rifampin, D-(+)-Trehalose, D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof. Compounds that may be used in the present invention include,for example, those compounds showing a greater inhibitory activity asshown in FIG. 4, such as, for example, Fluoroscein, Acridine Orange,Diamino Acridine, Crystal Violet, Thioflavin T, Oxytetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,(−)-Nicotine, Serotonin, Dopamine, Curcumin, Rifampin, D-(+)-Trehalose,D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof. Compounds that may be used in the present invention include,for example, those compounds showing a greater inhibitory activity asshown in FIG. 4, such as, for example, Fluoroscein, Acridine Orange,Diamino Acridine, Crystal Violet, Thioflavin T, Oxytetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,(−)-Nicotine, Dopamine, D-(+)-Trehalose, and derivatives and analogsthereof. Compounds that may be used in the present invention include,for example, those compounds showing a greater inhibitory activity asshown in FIG. 4, such as, for example, Fluoroscein, Acridine Orange,Diamino Acridine, Crystal Violet, Oxytetracycline, Chlortetracycline,Tannic Acid, (+)-Catechin, (−)-Nicotine, Dopamine, and derivatives andanalogs thereof. Compounds that may be used in the present inventioninclude, for example, those compounds showing a greater inhibitoryactivity as shown in FIG. 4, such as, for example, Fluoroscein, AcridineOrange, Crystal Violet, Oxytetracycline, (+)-Catechin, (−)-Nicotine,Dopamine, and derivatives and analogs thereof.

In other, illustrative examples of the present invention, compounds thatmay be used to prevent or alleviate the symptoms of a neuronal diseasesuch as, for example, Alzheimer's disease include, for example,compounds selected from the group consisting of Fluoroscein, DiaminoAcridine, Crystal Violet, Oxytetracycline, Tetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (÷)-Catechin,(−)-Nicotine, Morin, Serotonin, Dopamine, Curcumin, (R)-Ibuprofen,(S)-Naproxen, Rifampin, D-(+)-Trehalose, D-nicotine, pegylatedderivatives of 1-(p-aminophenyl)-6-methylbenzothiazole, and derivativesand analogs thereof. Compounds that may be used include, for example,those compounds showing a greater inhibitory activity as shown in FIG.4, such as, for example, compounds selected from the group consisting ofFluoroscein, Diamino Acridine, Crystal Violet, Oxytetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,(−)-Nicotine, Morin, Serotonin, Dopamine, Curcumin, (R)-Ibuprofen,Rifampin, D-(+)-Trehalose, D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof. Compounds that may be used include, for example, thosecompounds showing a greater inhibitory activity as shown in FIG. 4, suchas, for example, Fluoroscein, Diamino Acridine, Crystal Violet,Oxytetracycline, Chlortetracycline, Tannic Acid, Rosmarinic Acid,(+)-Catechin, (−)-Nicotine, Serotonin, Dopamine, Curcumin, Rifampin,D-(+)-Trehalose, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof. Compounds that may be used include, for example, thosecompounds showing a greater inhibitory activity as shown in FIG. 4, suchas, for example, Fluoroscein, Diamino Acridine, Crystal Violet,Oxytetracycline, Chlortetracycline, Tannic Acid, Rosmarinic Acid,(+)-Catechin, (−)-Nicotine, Dopamine, D-(+)-Trehalose, and derivativesand analogs thereof. Compounds that may be used include, for example,Fluoroscein, Crystal Violet, Oxytetracycline, Chlortetracycline, TannicAcid, Rosmarinic Acid, (+)-Catechin, (−)-Nicotine, Dopamine,D-(+)-Trehalose, and derivatives and analogs thereof. Compounds that maybe used include, for example, those compounds showing a greaterinhibitory activity as shown in FIG. 4, such as, for example,Fluoroscein, Diamino Acridine, Crystal Violet, Oxytetracycline,Chlortetracycline, Tannic Acid, (+)-Catechin, (−)-Nicotine, Dopamine,and derivatives and analogs thereof. Compounds that may be used include,for example, Fluoroscein, Crystal Violet, Oxytetracycline,Chlortetracycline, Tannic Acid, (+)-Catechin, (−)-Nicotine, Dopamine,and derivatives and analogs thereof. Compounds that may be used include,for example, those compounds showing a greater inhibitory activity asshown in FIG. 4, such as, for example, Fluoroscein, Crystal Violet,Oxytetracycline, (+)-Catechin, (−)-Nicotine, Dopamine, and derivativesand analogs thereof.

In yet other, illustrative examples of the present invention, compoundsthat may be used for diagnostic imaging of A-beta fibrils include, forexample, compounds selected from the group consisting of Fluoroscein,Diamino Acridine, Crystal Violet, Oxytetracycline, Tetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,(−)-Nicotine, Morin, Serotonin, Dopamine, Curcumin, (R)-Ibuprofen,(S)-Naproxen, Rifampin, D-(+)-Trehalose, D-nicotine, pegylatedderivatives of 1-(p-aminophenyl)-6-methylbenzothiazole, and derivativesand analogs thereof. Compounds that may be used include, for example,those compounds showing a greater inhibitory activity as shown in FIG.4, such as, for example, compounds selected from the group consisting ofFluoroscein, Diamino Acridine, Crystal Violet, Oxytetracycline,Chlortetracycline, Tannic Acid, Rosmarinic Acid, (+)-Catechin,(−)-Nicotine, Morin, Serotonin, Dopamine, Curcumin, (R)-Ibuprofen,Rifampin, D-(+)-Trehalose, D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof. Compounds that may be used include, for example, thosecompounds showing a greater inhibitory activity as shown in FIG. 4, suchas, for example, Fluoroscein, Diamino Acridine, Crystal Violet,Oxytetracycline, Chlortetracycline, Tannic Acid, Rosmarinic Acid,(+)-Catechin, (−)-Nicotine, Serotonin, Dopamine, Curcumin, Rifampin,D-(+)-Trehalose, D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof. Compounds that may be used include, for example, thosecompounds showing a greater inhibitory activity as shown in FIG. 4, suchas, for example, Fluoroscein, Diamino Acridine, Crystal Violet,Oxytetracycline, Chlortetracycline, Tannic Acid, Rosmarinic Acid,(+)-Catechin, (−)-Nicotine, Dopamine, D-(+)-Trehalose, and derivativesand analogs thereof. Compounds that may be used include, for example,Fluoroscein, Crystal Violet, Oxytetracycline, Chlortetracycline, TannicAcid, Rosmarinic Acid, (+)-Catechin, (−)-Nicotine, Dopamine,D-(+)-Trehalose, and derivatives and analogs thereof. Compounds that maybe used include, for example, those compounds showing a greaterinhibitory activity as shown in FIG. 4, such as, for example,Fluoroscein, Diamino Acridine, Crystal Violet, Oxytetracycline,Chlortetracycline, Tannic Acid, (+)-Catechin, (−)-Nicotine, Dopamine,and derivatives and analogs thereof. Compounds that may be used include,for example, Fluoroscein, Crystal Violet, Oxytetracycline,Chlortetracycline, Tannic Acid, (+)-Catechin, (−)-Nicotine, Dopamine,and derivatives and analogs thereof. Compounds that may be used include,for example, Fluoroscein, Crystal Violet, Oxytetracycline,Chlortetracycline, Tannic Acid, (+)-Catechin, (−)-Nicotine, Dopamine,and derivatives and analogs thereof. Compounds that may be used include,for example, those compounds showing a greater inhibitory activity asshown in FIG. 4, such as, for example, Fluoroscein, Crystal Violet,Oxytetracycline, (+)-Catechin, (−)-Nicotine, Dopamine, and derivativesand analogs thereof.

Compounds

Compounds of the present invention include, for example, those ofFormula I, including those listed as embodiments, exemplary embodiments,and examples, thereof. For example, compounds include compounds such asBTA-EG₄, BTA-EG₆, BTA-AZA-Crown₅, or BTA-EG₆-BTA.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom.

The compounds of the present invention, and compounds used in themethods of the present invention, may exist as salts. The presentinvention includes such salts. Examples of applicable salt forms includehydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates,(−)-tartrates or mixtures thereof including racemic mixtures,succinates, benzoates and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in art.Also included are base addition salts such as sodium, potassium,calcium, ammonium, organic amino, or magnesium salt, or a similar salt.When compounds of the present invention and compounds used in themethods of the present invention, contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples of acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived organic acids like acetic, propionic,isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like. Certain specificcompounds of the present invention and compounds used in the methods ofthe present invention, contain both basic and acidic functionalitiesthat allow the compounds to be converted into either base or acidaddition salts.

Certain compounds of the present invention, and compounds used in themethods of the present invention, can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are encompassed within thescope of the present invention. Certain compounds of the presentinvention and compounds used in the methods of the present invention,may exist in multiple crystalline or amorphous forms. In general, allphysical forms are equivalent for the uses contemplated by the presentinvention and are intended to be within the scope of the presentinvention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)-for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of the present invention and compounds used in the methodsof the present invention, may also contain unnatural proportions ofatomic isotopes at one or more of atoms that constitute such compounds.For example, the compounds may be radiolabeled with radioactiveisotopes, such as for example tritium (3H), iodine-125 (¹²⁵I) orcarbon-14 (¹⁴C). All isotopic variations of the compounds of the presentinvention, and compounds used in the methods of the present invention,whether radioactive or not, are encompassed within the scope of thepresent invention.

The compounds of the present invention may be synthesized using one ormore protecting groups generally known in the art of chemical synthesis.The term “protecting group” refers to chemical moieties that block someor all reactive moieties of a compound and prevent such moieties fromparticipating in chemical reactions until the protective group isremoved, for example, those moieties listed and described in Greene, etal., Protective Groups in Organic Synthesis, 3rd ed. John Wiley & Sons(1999). It may be advantageous, where different protecting groups areemployed, that each (different) protective group be removable by adifferent means. Protective groups that are cleaved under totallydisparate reaction conditions allow differential removal of suchprotecting groups. For example, protective groups can be removed byacid, base, and hydrogenolysis. Groups such as trityl, dimethoxytrityl,acetal and t-butyldimethylsilyl are acid labile and may be used toprotect carboxy and hydroxy reactive moieties in the presence of aminogroups protected with Cbz groups, which are removable by hydrogenolysis,and Fmoc groups, which are base labile. Carboxylic acid and hydroxyreactive moieties may be blocked with base labile groups such as,without limitation, methyl, ethyl, and acetyl in the presence of aminesblocked with acid labile groups such as t-butyl carbamate or withcarbamates that are both acid and base stable but hydrolyticallyremovable.

Carboxylic acid and hydroxy reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups capable of hydrogen bonding with acids may be blockedwith base labile groups such as Fmoc. Carboxylic acid reactive moietiesmay be blocked with oxidatively-removable protective groups such as2,4-dimethoxybenzyl, while co-existing amino groups may be blocked withfluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with apalladium(O)-catalyzed reaction in the presence of acid labile t-butylcarbamate or base-labile acetate amine protecting groups. Yet anotherform of protecting group is a resin to which a compound or intermediatemay be attached. As long as the residue is attached to the resin, thatfunctional group is blocked and cannot react. Once released from theresin, the functional group is available to react.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituent moieties found on thecompounds described herein. When compounds of the present invention andcompounds used in the methods of the present invention, containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention and compoundsused in the methods of the present invention, contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, for example, Bergeet al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977,66, 1-19). Certain specific compounds of the present invention containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

The terms “a,” “an,” or “a(n)”, when used in reference to a group ofsubstituents herein, mean at least one. For example, where a compound issubstituted with “an” alkyl or aryl, the compound is optionallysubstituted with at least one alkyl and/or at least one aryl. Moreover,where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different.

Description of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions.

Assays

Provided herein in the Examples are examples of screening assays thatutilize 96 well microtiter plates. It will be apparent to those ofordinary skill in the art that these assays may be adapted for othertypes of microtiter plates, including those made of various materialsand comprising various numbers of wells. Further, it is apparent tothose of ordinary skill in the art that these assays may be adapted toother high throughput methods, including other solid supports methodssuch as beads, microarrays, and stamping. (Mayer, M., et al.,Proteomics, 2004, 4:2366-76; G. MacBeath and S. L. Schreiber, Science,2000 289(5485): 1760-1763.)

The A-beta fibrils, A-beta fibrils pre-incubated with a test compound,or the detection reagent may, for example, be immobilized to a solidsupport. It is understood that immobilization can occur by any means,including for example; by covalent attachment, by electrostaticimmobilization, by attachment through a ligand/ligand interaction, bycontact or by depositing on the surface.

As used herein “solid support” or “solid carrier” means any solid phasematerial upon which an oligomer is synthesized, attached, ligated orotherwise immobilized. Solid support encompasses terms such as “resin”,“solid phase”, “surface” and “support”. A solid support may be composedof organic polymers such as polystyrene, polyethylene, polypropylene,polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well asco-polymers and grafts thereof. A solid support may also be inorganic,such as glass, silica, controlled-pore-glass (CPG), or reverse-phasesilica. The configuration of a solid support may be in the form ofbeads, spheres, particles, granules, a gel, or a surface. Surfaces maybe planar, substantially planar, or non-planar. Solid supports may beporous or non-porous, and may have swelling or non-swellingcharacteristics. A solid support may be configured in the form of awell, depression or other container, vessel, feature or location. Aplurality of solid supports may be configured in an array at variouslocations, addressable for robotic delivery of reagents, or by detectionmeans including scanning by laser illumination and confocal ordeflective light gathering.

Microarray or array means a predetermined spatial arrangement of samplespresent on a solid support or in an arrangement of vessels. Thesesamples may be, for example, A-beta fibrils, A-beta fibrilspre-incubated with test compounds, or may, for example, be secondbinding molecules or detection antibodies where, for example, the solidsupport is bound to the detection reagent, and the assay comprisesadding the preincubated A-beta fibrils to the second binding molecule.Certain array formats are referred to as a “chip” or “biochip” (M.Schena, Ed. Microarray Biochip Technology, BioTechnique Books, EatonPublishing, Natick, Mass. (2000). An array can comprise a low-densitynumber of addressable locations, e.g. 2 to about 12, medium-density,e.g. about a hundred or more locations, or a high-density number, e.g. athousand or more. Typically, the array format is a geometrically regularshape that allows for fabrication, handling, placement, stacking,reagent introduction, detection, and/or storage. The array may beconfigured in a row and column format, with regular spacing between eachlocation. Alternatively, the locations may be bundled, mixed orhomogeneously blended for equalized treatment or sampling. An array maycomprise a plurality of addressable locations configured so that eachlocation is spatially addressable for high-throughput handling, roboticdelivery, masking, or sampling of reagents, or by detection meansincluding scanning by laser illumination and confocal or deflectivelight gathering.

The presence of a compound that blocks the binding of a second bindingmolecule to A-beta fibrils is generally detected using a second bindingmolecule that binds to A-beta fibrils. The second binding molecule iseither directly labeled, i.e., comprise or reacts to produce adetectable label, or is indirectly labeled, i.e., bind to a moleculecomprising or reacting to produce a detectable label. Labels can bedirectly attached to or incorporated into the detection reagent bychemical or recombinant methods.

In one embodiment, the detection reagent, the molecule that is detectedin the screening assay, is a second binding molecule that is an antibodythat specifically binds to A-beta peptide. In another embodiment, thedetection reagent is an antibody that specifically binds to the secondbinding molecule. In one embodiment, a label is coupled to the detectionreagent through a chemical linker. Linker domains are typicallypolypeptide sequences, such as poly gly sequences of between about 5 and200 amino acids. In some embodiments, proline residues are incorporatedinto the linker to prevent the formation of significant secondarystructural elements by the linker. Preferred linkers are often flexibleamino acid subsequences which are synthesized as part of a recombinantfusion protein comprising the RNA recognition domain. In one embodiment,the flexible linker is an amino acid subsequence that includes aproline, such as Gly(x)-Pro-Gly(x) where x is a number between about 3and about 100. In other embodiments, a chemical linker is used toconnect synthetically or recombinantly produced recognition and labelingdomain subsequences. Such flexible linkers are known to persons of skillin the art. For example, poly(ethylene glycol) linkers are availablefrom Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionallyhave amide linkages, sulfhydryl linkages, or heterofunctional linkages.

The detectable labels used in the assays of the present invention, whichare attached to the detection reagent, can be primary labels (where thelabel comprises an element that is detected directly or that produces adirectly detectable element) or secondary labels (where the detectedlabel binds to a primary label, e.g., as is common in immunologicallabeling). An introduction to labels, labeling procedures and detectionof labels is found in Polak and Van Noorden (1997) Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, N.Y. and in Haugland(1996) Handbook of Fluorescent Probes and Research Chemicals, a combinedhandbook and catalogue Published by Molecular Probes, Inc., Eugene,Oreg. Patents that described the use of such labels include U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149;and 4,366,241.

Primary and secondary labels can include undetected elements as well asdetected elements. Useful primary and secondary labels in the presentinvention can include spectral labels such as green fluorescent protein,fluorescent dyes (e.g., fluorescein and derivatives such as fluoresceinisothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives(e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.),digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase etc.), spectralcalorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads. The label can becoupled directly or indirectly to a component of the detection assay(e.g., the detection reagent) according to methods well known in theart. As indicated above, a wide variety of labels may be used, with thechoice of label depending on sensitivity required, ease of conjugationwith the compound, stability requirements, available instrumentation,and disposal provisions.

Preferred labels include those that use: 1) chemiluminescence (usinghorseradish peroxidase and/or alkaline phosphatase with substrates thatproduce photons as breakdown products as described above) with kitsbeing available, e.g., from Molecular Probes, Amersham,Boehringer-Mannheim, and Life Technologies/Gibco BRL; 2) colorproduction (using both horseradish peroxidase and/or alkalinephosphatase with substrates that produce a colored precipitate (kitsavailable from Life Technologies/Gibco BRL, and Boehringer-Mannheim));3) fluorescence using, e.g., an enzyme such as alkaline phosphatase,together with the substrate AttoPhos (Amersham) or other substrates thatproduce fluorescent products, 4) fluorescence (e.g., using Cy-5(Amersham), fluorescein, and other fluorescent tags); 5) radioactivity.Other methods for labeling and detection will be readily apparent to oneskilled in the art.

For use of the present invention in the clinic, preferred labels arenon-radioactive and readily detected without the necessity ofsophisticated instrumentation. Preferably, detection of the labels willyield a visible signal that is immediately discernable upon visualinspection. One preferred example of detectable secondary labelingstrategies uses an antibody that recognizes A-beta amyloid fibrils inwhich the antibody is linked to an enzyme (typically by recombinant orcovalent chemical bonding). The antibody is detected when the enzymereacts with its substrate, producing a detectable product. Preferredenzymes that can be conjugated to detection reagents of the inventioninclude, e.g., β-galactosidase, luciferase, horse radish peroxidase, andalkaline phosphatase. The chemiluminescent substrate for luciferase isluciferin. One embodiment of a fluorescent substrate for β-galactosidaseis 4-methylumbelliferyl-β-D-galactoside. Embodiments of alkalinephosphatase substrates include p-nitrophenyl phosphate (pNPP), which isdetected with a spectrophotometer; 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) and fast red/napthol AS-TRphosphate, which are detected visually; and4-methoxy-4-(3-phosphonophenyl) spiro[1,2-dioxetane-3,2′-adamantane],which is detected with a luminometer. Embodiments of horse radishperoxidase substrates include 2,2′azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS), 5-aminosalicylic acid (5AS), o-dianisidine, ando-phenylenediamine (OPD), which are detected with a spectrophotometer,and 3,3,5,5′-tetramethylbenzidine (TMB), 3,3′ diaminobenzidine (DAB),3-amino-9-ethylcarbazole (AEC), and 4-chloro-1-naphthol (4C1N), whichare detected visually. Other suitable substrates are known to thoseskilled in the art. The enzyme-substrate reaction and product detectionare performed according to standard procedures known to those skilled inthe art and kits for performing enzyme immunoassays are available asdescribed above.

The presence of a label can be detected by inspection, or a detectorwhich monitors a particular probe or probe combination is used to detectthe detection reagent label. Typical detectors includespectrophotometers, phototubes and photodiodes, microscopes,scintillation counters, cameras, film and the like, as well ascombinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons ofskill. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

Research Reagents

The present invention is further directed to research reagents used todetect amyloid proteins and amyloid plaques. Such research reagents arecompounds that bind to amyloid proteins, including, for example, but notlimited to, the compounds of the present invention. Research reagents ofthe present invention may further comprise dyes or other detectablelabels. Thus, research reagents of the present invention include, forexample, compositions that comprise the compounds of the presentinvention, and compounds of the present invention.

The research reagents may be used, for example, to detect the presenceof amyloid plaques in vivo, in tissues, in cells, and in tissue or cellextracts. The research reagents may be used, for example, to determinethe existence of an amyloid-associated disease, or to assist inscreening for compounds that may prevent or alleviate the symptoms ofthe disease. The research reagents may be used, for example, to inhibitthe interaction of an amyloid protein with a second binding protein,thus enabling the study of a cellular or disease mechanism. In oneexemplary embodiment, a method is provided for detecting the presence ofan amyloid protein, or an amyloid plaque, comprising contacting saidamyloid protein or amyloid plaque with a research reagent of the presentinvention, and detecting binding of the research reagent to the amyloidprotein or amyloid plaque.

Kits

The present invention is also directed to kits that utilize thescreening assays described herein. A basic kit for measuring thepresence and/or activity of an A-beta fibril binding molecule includes avessel or surface on which A-beta fibril molecules can bind, A-betafibril molecules, and an anti-A-beta antibody. Reagents used to detectthe binding of the anti-A-beta antibody, such as reagents used for ELISAassays may also be included in the kit. The kit may also compriseinstructions for use of the kit. Rather than including an anti-A-betaantibody, the kit may include other detectable molecules that are knownto bind A-beta fibril molecules. The kit may also include an appropriateamount of reaction buffer disposed in a suitable container.

“Instructions for use,” is a tangible expression describing the reagentconcentration or at least one assay method parameter such as therelative amount of reagent and sample to be admixed, maintenance timeperiods for reagent/sample admixtures, temperature, buffer conditions,and the like. The instructions for use are suitable to enable an analystto carry out the desired assay.

In some embodiments, the kits can include a container containing A-betafibrils, either free or bound to solid supports. Also included in thekits can be a suitable membrane, solid support, or vessel used inconducting the assay such as, for example, a microtiter plate, forexample, but not limited to, a polystyrene plate. Preferably, the kitswill also include reagents used in the described assays, includingreagents useful for detecting the binding of a compound to the fibrils,such as, for example, a detectable molecule that binds to A-betafibrils. For example, the kits may include an anti-A-beta antibody. Theantibody itself may have a label, such as a flourescein label or dye,that may allow it to be detected. Other materials useful in theperformance of the assays can also be included in the kits, includingtest tubes, transfer pipettes, and the like. The kits can also includewritten instructions for the use of one or more of these reagents in anyof the assays described herein.

The kits of the invention can also include an internal and/or anexternal control. The control can consist of, for example, Congo Red, aCongo Red derivative, ThT or a ThT derivative. The control may, forexample, be selected from the group consisting of Congo Red,Fluoroscein, Acridine Orange, Diamino Acridine, Crystal Violet,Thioflavin T, Oxytetracycline, Tetracycline, Chlortetracycline, TannicAcid, Rosmarinic Acid, (+)-Catechin, (−)-Nicotine, Morin, Serotonin,Dopamine, Curcumin, (R)-Ibuprofen, (S)-Naproxen, Rifampin,D-(+)-Trehalose, D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and derivatives and analogsthereof. The kits of the present invention can contain materialssufficient for one assay, or can contain sufficient materials formultiple assays.

Also included in the scope of the present invention are kits that may beused to detect amyloid proteins or amyloid plaques, wherein such kitscomprise research reagents of the present invention. A basic kit fordetecting or measuring the quantity of amyloid protein or an amyloidplaque includes at least one research reagent of the present invention.Reagents used to detect the binding of the research reagent, such asdyes, labels, antibodies, and the like, may also be included in the kit.The kit may also comprise instructions for use of the kit. The kit mayalso include an appropriate amount of reaction buffer disposed in asuitable container.

“Instructions for use,” is a tangible expression describing the reagentconcentration or at least one assay method parameter such as therelative amount of reagent and sample to be admixed, maintenance timeperiods for reagent/sample admixtures, temperature, buffer conditions,and the like. The instructions for use are suitable to enable an analystto carry out the desired assay.

In some embodiments, the kits can include a container containing A-betafibrils, either free or bound to solid supports, as a positive control.Or, the kits may include a container containing amyloid plaques as apositive control. Also included in the kits can be a suitable membrane,solid support, or vessel used in conducting the assay such as, forexample, a microtiter plate, for example, but not limited to, apolystyrene plate. Preferably, the kits will also include reagents usedin the described assays, including reagents useful for detecting thebinding of a research reagent to the fibrils, such as, for example, adetectable molecule that binds to research reagent/A-beta fibrilcomplexes. Other materials useful in the performance of the assays canalso be included in the kits, including test tubes, transfer pipettes,and the like. The kits can also include written instructions for the useof one or more of these reagents in any of the assays described herein.

The amounts of the various reagents in the kits can be varied dependingon various factors, such as the optimum sensitivity of the assay, thenumber of assays to be performed, etc.

It is within the scope of the invention to provide manual test kits ortest kits for use in automated analyzers.

Formulation

While the compounds of the present invention will typically be used intherapy for human patients, they may also be used in veterinary medicineto treat similar or identical diseases. The compounds of the presentinvention and compounds used in the methods of the present invention,include geometric and optical isomers.

The compounds according to the invention are effective over a widedosage range. For example, in the treatment of adult humans, dosagesfrom 0.01 to 1000 mg, from 0.02 to 800 mg, from 0.05 to 700 mg, from 0.1to 650 mg, from 0.2 to 600 mg, from 0.5 to 500 mg, from 0.5 to 300 mg,from 0.5 to 250 mg, 0.5 to 100 mg, from 1 to 100 mg, from 1 to 50 mg,and from 1 to 50 mg per day, from 5 to 40 mg per day are examples ofdosages that may be used. One example of a dosage is 10 to 30 mg perday. The exact dosage will depend upon the route of administration, theform in which the compound is administered, the subject to be treated,the body weight of the subject to be treated, and the preference andexperience of the attending physician.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art and may include, by way of example but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington: The Science and Practice of Pharmacy (20 th ed.) Lippincott,Williams & Wilkins (2000). Preferred pharmaceutically acceptable saltsinclude, for example, acetate, benzoate, bromide, carbonate, citrate,gluconate, hydrobromide, hydrochloride, maleate, mesylate, napsylate,pamoate (embonate), phosphate, salicylate, succinate, sulfate, ortartrate.

In therapeutic and/or diagnostic applications, the compounds of theinvention may be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington: The Science andPractice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-low release form as is known to those skilled in theart. Techniques for formulation and administration may be found inRemington: The Science and Practice of Pharmacy (20th ed.) Lippincott,Williams & Wilkins (2000). Suitable routes may include oral, buccal,sublingual, rectal, transdermal, vaginal, transmucosal, nasal orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

In illustrative embodiments, for injection, such as, for example,intravenous delivery, the agents of the invention may be formulated inaqueous solutions, such as in physiologically compatible buffers such asHank's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. Use of pharmaceutically acceptable carriersto formulate the compounds herein disclosed for the practice of theinvention into dosages suitable for systemic administration, or fortargeted administration, such as that targeted to the brain, is withinthe scope of the invention. With proper choice of carrier and suitablemanufacturing practice, the compositions of the present invention, inparticular, those formulated as solutions, may be administeredparenterally, such as by intravenous injection. The compounds may beformulated readily using pharmaceutically acceptable carriers well knownin the art into dosages suitable for oral administration. Such carriersenable the compounds of the invention to be formulated as tablets,pills, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprising oneor more buffers, excipients, salts, preservative, auxiliaries and thelike which facilitate processing of the active compounds intopreparations which may be used pharmaceutically. The preparationsformulated for oral administration may be in the form of tablets,dragees, capsules, or solutions. Appropriate pharmaceutically acceptablecarriers are known to those of ordinary skill in the art and may befound in, for example, Remington: The Science and Practice of Pharmacy(20th ed.) Lippincott, Williams & Wilkins (2000).

Pharmaceutical preparations for oral use may be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that may be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

Pharmaceutical compositions of the present invention are those that, inaddition to specifically binding amyloid in vivo and capable of crossingthe blood brain barrier, are also non-toxic at appropriate dosage levelsand have a satisfactory duration of effect.

EXAMPLES

All synthetic reagents were from Aldrich, Fisher Scientific, Alfa Aesaror Fluka, and were used as received. Water was filtered through aNANOPure Diamond™ water purification system from Barnstead (18.2 μΩ/cm).Aβ-peptide (1-42) was obtained from Biopeptide Co, LLC, San Diego,Calif.; 96-well plates from Nalge Nunc International, Rochester, N.Y.;catalase from human erythrocytes (Lot #B67459) from Calbiochem, SanDiego, Calif.; IgGs from Abcam, Cambridge, Mass.; and bovine serumalbumin (BSA, fraction V) from Omni Pur.

NMR spectra were obtained on a Varian 400 MHz spectrometer. Chemicalshifts are reported in ppm relative to residual solvent. FT-IR spectrawere obtained on a Nicholet MAGNA-IR 440 spectrometer. A Perkin ElmerHTS-7000 Bio Assay reader was used to measure the absorbance of, theassays. UV-Vis absorbencies were determined with a Beckman-Coulter DU500spectrometer.

Example 1

A-beta fibrils were grown in vitro from synthetic AD-related A-betapeptides (residues 1-42). Fibrils were characterized by atomic forcemicroscopy. Images indicated the presence of fibrils that wereconsistent with literature reports (Hilbich, C., et al., J. Mol. Biol.1992, 228: 460.) in terms of size (5-10 nm in diameter and >400 nm long)and in terms of morphology (single fibrils and bundles of fibrils). Thewells of commercial 96-well plates were coated with freshly preparedA-beta fibrils and the fibrils were incubated with solutions of ThT.After removal of excess ThT, the ThT-coated fibrils in the wells weretreated with a monoclonal anti-A-beta IgG (clone 6E10, derived fromresidues 3-8 of A-beta peptide as antigens). The interaction of theanti-A-beta IgG with the ThT-coated A-beta fibrils was quantified usingan ELISA-based assay.

FIG. 2 a shows that ThT had an inhibition concentration corresponding to50 percent inhibition (IC₅₀) of 5 μM for the binding of the anti-A-betaIgG (clone 6E10, 0.16 μg mL⁻¹) to the A-beta fibrils (deposited fromsolutions containing 1.3 μM A-beta peptide). Because the finalconcentration of the A-beta fibrils deposited in the wells was notdetermined, the A-beta fibrils (1.3 μM) were also incubated withsolutions of ThT prior to depositing the coated fibers into the wellsand an IC₅₀ of 60 μM was measured (See Example 3, FIG. 3). Forcomparison, an IC₅₀ of ˜1 μM was observed when a 0.3 μM solution ofA-beta peptide was used in the control procedure (data not shown).Therefore, it would be expected that observed IC₅₀'s would be differentusing the two procedures. A total inhibition of 65% of the interactionbetween this IgG and A-beta fibrils was measured when the fibrils wereincubated with a 50 μM solution of ThT. 0 percent inhibition was definedas the UV-Vis signal observed when the assay is run in the absence ofThT and 100 percent inhibition was defined as the UV-Vis signal observedwhen the assay is run in the absence of both amyloid fibril and ThT.Solutions of ThT with concentrations higher than 50 μM did not increasethe total inhibition of the IgG-A-beta fibril interactions above 65%.Coating the fibrils in solutions of ThT (>100 μM of Tyr) prior todeposition into wells resulted in a maximum inhibition of ˜80% of theprotein-amyloid interactions (See Example 3, FIG. 3). Exposing theThT-coated A-beta fibrils to prolonged washing steps (from 0-4 hrs) withPBS buffer prior to incubation with primary anti-A-beta IgG did notaffect the total amount of inhibition of the IgG-amyloid interactions,suggesting the rate of unbinding of ThT from the A-beta fibrils is slowrelative to the timescale of the binding assay.

Example 2

To demonstrate that this surface-coatings approach can extend to otherproteins that bind to A-beta fibrils, the ability of ThT to inhibit theinteraction of A-beta fibrils with an anti-A-beta IgG raised against adifferent epitope of A-beta peptide (clone AMY-33, derived from residues1-28 of A-beta peptide as antigens) was tested. An IC₅₀ of 0.4 μM (FIG.2 b) for ThT with this IgG (clone AMY-33) was measured under the sameconditions used to assay the first anti-A-beta IgG (clone 6E10). Amaximum inhibition of ˜65% of this IgG (clone MY-33)-A-beta fibrilinteraction was measured with solutions of ThT having concentrations of10 μM or higher. No inhibition of the interaction between the IgGs andA-beta fibrils in control experiments using I-naphthol-4-sulfonate (FIG.2 c) were observed. Structurally-similar molecules to1-naphthol-4-sulfonate do not interact with A-beta fibrils. (Villa, S.,et al., Farmaco 2003, 58: 929.) This may suggest that binding of thesmall molecule to the A-beta fibrils is necessary for the observedinhibition with ThT.

Without limitation as to the possible mechanism of action of inhibition,the observed partial inhibition of the IgG-amyloid interactions by ThTmay be, for example, due to ThT not binding (or binding differently) tothe terminal ends of the A-beta fibrils (ThT is known to bind only tothe fibril form of A-beta peptides (LeVine III, H. Arch. Biochem.Biophys. 1997, 342: 306)). It is possible, therefore, that about 35% ofthe surface area of each A-beta fibril (presumably localized near theends of A-beta fibril) may still be accessible for binding byanti-A-beta IgGs even after coating the surface of A-beta fibrils withThT. Perhaps molecules that more thoroughly coat the surface of A-betafibrils compared to ThT may show increased inhibition of protein-A-betafibril interactions.

These examples demonstrate that ThT can inhibit 65±10% of IgG-A-betafibril interactions. The generation of protein-resistive surfacecoatings on amyloid fibrils with small molecules may lead to newtherapeutic strategies for the inhibition of harmful protein-amyloidinteractions in neurodegenerative diseases.

ADDITIONAL REFERENCES

-   Yan, S. D. et al. Nature 1997, 389, 689: Yan, S. D.; Fu, J.; Sot,    C.; Chen, X.; Zhu, H.; Al-Mohanna, F.; Collison, K.; Zhu, A.; Stem,    E.; Saido, T.; Tohyama, M.; Ogawa, S.; Roher, A.; Stern, D. Nature    1997, 389, 689:-   Yan, S. D. et al. J. Biol. Chem. 1999, 274, 2145: Yan, S. D.; Shi,    Y.; Zhu, A.; Fu, J.; Zhu, H.; Zhu, Y.; Gibson, L; Stem, E.;    Collison, K.; Al-Mohanna, F.; Ogawa, S.; Roher, A.; Clarke, S. G.;    Stem, D. M. J. Biol. Chem. 1999, 274, 2145.-   Lustbader, J. W. et al. Science 2004, 304, 448: Lustbader, J. W.;    Girilli, M.; Lin, C.; Xu, H. W.; Takuma, K.; Wang, N.; Caspersen,    C.; Chen, X.; Pollak, S.; Chaney, M.; Trinchese, F.; Liu, S.;    Gunn-Moore, F.; Lue, L. F.; Walker, D. G.; Kuppusamy, P:; Zewier, Z.    L.; Aranchio, O.; Stem, D.; Yan, S. S. D.; Wu, H. Science 2004, 304,    448.-   Yan, S. D. et al. Nature 1996, 382, 685: Yan, S. D.; Chen, X.; Fu,    J.; Chen, M.; Zhu, H.; Roher, A.; Slattery, T.; Zhao, L.; Nagashima,    M.; Morser, J.; Migheli, A.; Nawroth, P.; Stem, D.; Schmidt, A. M.    Nature 1996, 382, 685.

Example 3 Assay Methods

A-beta fibrils were grown from synthetic A-beta (142) peptides(Biopeptide Co, LLC, San Diego, Calif., USA) by dissolving 30 μg ofpeptide in 90 μL of water and incubating at 37° C. for 72 hours. Fibrilswere characterized by atomic force microscopy. Images indicated thepresence of fibrils (FIG. 1 a) that were consistent with literaturereports in terms of size (5-10 nm in diameter and >400 nm long) and interms of morphology (single fibrils and bundles of fibrils).

The wells of commercial 96-well plates were coated with freshly preparedA-beta fibrils. Each well of a 96 well plate (Titertek®, Huntsville,Ala., USA) was coated for 12 hours with 50 μL of a 5.8 μg/mL (1.3 μM)solution of A-beta peptides (present in fibril form) in phosphatebuffered saline (PBS, 10 mM NaH₂PO₄/Na₂HPO₄, 138 mM NaCl, 2.7 mM KCl,pH=7.4). After removal of the excess sample, 50 μl, of thioflavin T or1-naphthol-4-sulfonate solutions in PBS buffer (various concentrationswere obtained by diluting a stock solution with PBS buffer) wereincubated in the wells for 1.5 or 12 hours, followed by removal of theexcess solutions. Alternatively, amyloid fibrils were preincubated for1.5 hours with ThT at various concentrations by adding amyloid fibrils(having a final concentration of 1.3 μg/mL or 5.8 μg/mL) to the ThTsolutions. Wells were coated with ThT-bound fibrils by addition of 50 μLof the preincubated solutions per well and incubation for 1.5 hours.Excess solutions were then discarded. To test if the bound Thioflavin Twill be removed during the process of the assay, we subjected the sampleto extensive washing steps: 300 μL of PBS was added to the wells andequilibrated for 15 minutes, removed, and then repeated as many as 16times. The following steps were identical for both methods: All wellswere blocked for 30 minutes by adding 300 μL of a 1 (w/v) % solution ofbovine serum albumin (BSA, Fraction V, OmniPur) in PBS buffer. Wellswere washed once with 300 μL of PBS buffer and incubated for anadditional 1 hour with 50 μL of a 0.16 μg/mL or 0.5 μg/mL of anti-A-betaIgG (dilution 1:6000 in BSA/PBS for clone 6E10 or dilution 1:1000 in 1%BSA/PBS for clone AMY-33, respectively). The wells were washed twicewith 300 μL PBS buffer and incubated for 45 minutes with 50 μL of thesecondary IgG (1 μg/mL, dilution 1:1000 in 1% BSA/PBS), and washed twicewith 300 μL PBS buffer. Bound secondary IgGs were detected by theaddition of 50 μL of a p-nitrophenyl phosphate solution (1 mg/mL in 0.1Mdiethanol amine/0.5 mM magnesium chloride). After the desiredintensities were achieved, the enzymatic reaction was quenched after0.5-2 hours by the addition of 50 μL of a 0.25N sodium hydroxidesolution. Absorbance intensities were determined at 405 nm using aUV-Vis spectroscopic plate reader (HTS 7000 Bio Assay Reader, PerkinElmer, Fremont, Calif., USA). Each run was performed five times andaveraged. Graphs were plotted and fitted with the sigmoidal curvefitting option in Origin 6.0 (Microcal Software, Inc., Northhampton,Mass., USA).

Sodium chloride and sodium dihydrogen phosphate hydrate were purchasedfrom Fisher Scientific. Potassium chloride and sodium hydroxide werepurchased from Baker. Magnesium chloride was purchased from Sigma.Diethanolamine, p-nitrophenyl phosphate, and 1-naphthol-4-sulfonic acid(sodium salt) were purchased from Fluka. Thioflavin T (ThT) waspurchased from MP Biomedica. All reagents were used without furtherpurification. Water (18.2 μΩ/cm) was filtered through a NANOPureDiamond™ (Barnstead) water purification system before use. MetrologyProbe™, Tap 300 (Ted Pella, Inc, Redding, Calif., USA) probe tips wereused for AFM measurements.

As primary IgGs against A-beta, monoclonal anti-A-beta IgG (clone 6E10,mouse, derived from residues 3-8 of A-beta peptide as antigens,) wasobtained from Abeam, Cambridge, Mass., (Lot # 79040) and anti-A-beta IgG(clone AMY-33, mouse, derived from residues 1-28 of A-beta peptide asantigens,) was purchased from Zymed Laboratories Inc, South SanFrancisco, Calif., (Lot # 40487378). The secondary anti-mouse IgG(anti-mouse IgG H+L conjugated with alkaline phosphatase, polyclonal,from rabbit) was purchased from Abeam, Cambridge, Mass., (Lot # 71496 or#95504). All ELISA based procedures were done at 25° C. unless otherwisestated.

For imaging of A-beta fibrils by atomic force microscopy, 10 μL of anA-beta solution in distilled water (0.33 mg/mL) was placed on freshlycleaved mica (SPI, Westchester, Pa., USA) for 2 minutes. The solutionwas wicked off with filter paper and the sample was washed twice with 10μL of water. The sample was then dried under vacuum and imaged using aDI Nanoscope-IV Multimode AFM (Veeco, Santa Barbara, Calif., USA) intapping mode under ambient conditions.

Example 4 Compound Assays

The compounds shown in FIG. 4 were assayed using the A-beta fibril assayof Example 3 as follows.

A-beta fibrils were grown from synthetic A-beta (1-42) peptides(Biopeptide Co, LLC, San Diego, Calif., USA) by dissolving 30 μg ofpeptide in 90 μL of water and incubating at 37° C. for 72 hours. Fibrilswere characterized by atomic force microscopy. Images indicated thepresence of fibrils (FIG. 1 a) that were consistent with literaturereports in terms of size (5-10 nm in diameter and >400 nm long) and interms of morphology (single fibrils and bundles of fibrils).

The wells of commercial 96-well plates were coated with freshly preparedA-beta fibrils. Each well of a 96 well plate (Nalge Nunc, Rochester,N.Y.) was coated for 2 hours with 50 μL of a 5.8 μg/mL (1.3 μM) solutionof A-beta peptides (present in fibril form) in phosphate buffered saline(PBS, 10 mM NaH₂PO₄/Na₂HPO₄, 138 mM NaCl, 2.7 mM KCl, pH=7.4). Afterremoval of the excess sample, 50 μL of inhibitor solutions in PBS or 5%DMSO in PBS buffer (various concentrations were obtained by diluting astock solution with PBS buffer or the DMSO/PBS buffer solution; the DMSOwas added to assist in the solubility of inhibitors that were insolublein PBS buffer) were incubated in the wells for 12 hours, followed byremoval of the excess solutions. AU wells were blocked for 30 minutes byadding 300 μL of a 1 (w/v) % solution of bovine serum albumin (BSA,Fraction V, OmniPur) in PBS buffer. Wells were washed once with 300 μLof PBS buffer and incubated for an additional 1 hour with 50 μL of a0.16 μg/mL or 0.5 μg/mL of anti-A-beta IgG (dilution 1:6000 in BSA/PBSfor clone 6E10). The wells were washed twice with 300 μL PBS buffer andincubated for 45 minutes with 50 μL of the secondary IgG (1 μg/mL,dilution 1:1000 in 1% BSA/PBS), and washed twice with 300 μL PBS buffer.Bound secondary IgGs were detected by the addition of 50 μL of ap-nitrophenyl phosphate solution (1 mg/mL in 0.1M diethanol amine/0.5 mMmagnesium chloride). The enzymatic reaction was quenched after 0.5-2hours by the addition of 50 μL of a 0.25N sodium hydroxide solution.Absorbance intensities were determined at 405 nm using a UV-Visspectroscopic plate reader (HTS 7000 Bio Assay Reader, Perkin Elmer,Fremont, Calif., USA). Each run was performed five times and averaged.Graphs were plotted and fitted with the sigmoidal curve fitting optionin Origin 6.0 (Microcal Software, Inc., Northhampton, Mass., USA).

Sodium chloride and sodium dihydrogen phosphate hydrate were purchasedfrom Fisher Scientific. Potassium chloride and sodium hydroxide werepurchased from Baker. Magnesium chloride was purchased from Sigma.Diethanolamine, p-nitrophenyl phosphate was purchased from Fluka.Inhibitors were from MP Biomedical, Sigma, Aldrich, or Fluka. Allreagents were used without further purification. Water (18.2 μΩ/cm) wasfiltered through a NANOPure Diamond™ (Barnstead) water purificationsystem before use. Metrology Probe™, Tap 300 (Ted Pella, Inc, Redding,Calif., USA) probe tips were used for AFM measurements.

As primary IgGs against A-beta, monoclonal anti-A-beta IgG (clone 6E10,mouse, derived from residues 3-8 of A-beta peptide as antigens,) wasobtained from Abcam, Cambridge, Mass., (Lot # 79040 and 116274) Thesecondary anti-mouse IgG (anti-mouse IgG H+L conjugated with alkalinephosphatase, polyclonal, from rabbit) was purchased from Abcam,Cambridge, Mass., (Lot #95504). All ELISA based procedures were done at25° C.

The assay results are shown in the Table of FIG. 4, in which percentmaximum inhibition, μM maximum inhibition, and IC₅₀s are shown. Notshown in the table, but also showing good efficacy were D-nicotine andoligoethylene glycol derivatives of2-(p-aminophenyl)-6-methylbenzothiazole.

Example 5 Binding of THT Derivatives to A-Beta Fibrils

Molecules were assayed for their ability to bind with higher density toA-beta fibrils compared to ThT, to test whether these molecules wouldgenerate more complete protein-resistive coatings on A-beta fibrils.Substituted 2-(4-aminophenyl)-benzothiazoles (commonly referred to as“benzothiazole-aniline” or BTA) have been reported as biocompatibleanalogues of ThT(Klunk, W. E., et al., Life Sci. 2001, 69(13),1471-1484; a) C. Solbach, M. Uebele, G. Reischl, H.-J. Machulla, Appl.Radiation Isotope 2005, 62(4), 591-595; b) Y. Wang, W. E. Klunk, G.-F.Huang, M. L. Debnath, D. P. Holt, C. A. Mathis, J. Mol. Neurosci. 2002,19(1/2), 11-16; c) W. E. Klunk, Y. Wang, G.-f. Huang, M. L. Debnath, D.P. Holt, L. Shao, R. L. Hamilton, M. D. Ikonomovic, S. T. DeKosky, C. A.Mathis, J. Neurosci. 2003, 23(6), 2086-2092; C. A. Mathis, Y. Wang, W.E. Klunk, Curr. Pharm. Design 2004, 10(13), 1469-1492), that bind tomultiple sites along the A-beta fibril axis. (A. Lockhart, L. Ye, D. B.Judd, A. T. Merritt, P. N. Lowe, J. L. Morgenstern, G. Hong, A. D. Gee,J. Brown, J. Biol. Chem. 2005, 280(9), 7677-7684). BTA-EG₄ and BTA-EG₆(FIG. 1 b) were synthesized and tested to determine whether theseuncharged derivatives of ThT could also generate protein-resistivecoatings on A-beta fibrils.

Oligoethylene glycol derivatives of ThT were tested for their ability toinhibit the binding of a monoclonal anti-Aβ IgG (clone 6E10) to ADfibrils formed from residues 1-42 of synthetic A-beta peptides (FIG. 5).

BTA-EG₄ and BTA-EG₆ inhibited (0 percent inhibition was defined as theUV-Vis signal observed when the assay is run in the absence of smallmolecule and define 100 percent inhibition as the UV-Vis signal observedwhen the assay is run in the absence of both amyloid fibrils and smallmolecule.) 94% and 99%, respectively, of the IgG-A-beta fibrilinteractions, with inhibitory concentrations corresponding to 50%inhibition (IC₅₀'s) of ˜100 μM. These results suggest that the two BTAderivatives form almost complete IgG-resistive coatings on A-betafibrils.

Example 6 Binding Competition to A-Beta Fibrils

To test whether the derivatives of BTA could also inhibit biologicallyrelevant protein-amyloid interactions, we investigated whether BTA-EG₄and BTA-EG₆ could compete with two natural cellular proteins—catalaseand ABAD—for binding to A-beta fibrils. Catalase and ABAD were used asnatural proteins in these studies due to their previously reportedinteraction with aggregated A-beta and their potential importance in thepathogenesis of Alzheimer's disease. (Milton, N. G. N., et al.,Neuroreport 2001, 12(11), 2561-2566; Milton, N. G. N., et all, Biochem.J. 1999, 344(2), 293-296; Yan, S. D., et al., Int. J. Exp. Path. 2005,86(3), 161-171; Kissinger, C. N., et al., J. Mol. Biol. 2004, 342(3),943-952; Lustbader, J. W., et al., Science 2004, 304(5669), p 448-452).Catalase and ABAD have also been reported to bind to different residuesof A-beta (i.e., at residues 25-35 for catalase (Milton, et al.,Neuroreport 2001, 12:2561-2566) and residues 1-20 for ABAD (Oppermann,U. C. T., et al., FEBS Lett. 1999, 451(3), 238-242; Powell, A. J., etal., J. Mol. Biol. 2000, 303(2), 311-327). The different sites forbinding of these proteins to A-beta peptides provide an opportunity totest whether the oligoethylene glycol derivatives of BTA may havepotential general utility for inhibiting the interaction between A-betafibrils and proteins that bind to completely different regions ofA-beta.

Catalase is a peroxidase enzyme that catalyzes the breakdown of H₂O₂ inperoxisomes (Wiemer, E. A. C., et al, J. Immunol. Methods 1992,151(1-2), 165-175) and the cytoplasm (R. J. Marttila, M. Roytta, H.Lorentz, U. K. Rinne, J. Neural. Transm. 1988, 74(2), 87-95) and plays acentral role in suppressing the level of reactive oxygen species incells. (C. D. Putnam, A. S. Arvai, Y. Bourne, J. A. Tainer, J. Mol.Biol. 2000, 296(1), 295-309). Catalase has been found to be associatedwith Alzheimer's plaques (M. A. Lovell, W. D. Ehmann, S. M. P. Butler,W. R. Markesbery, Neurol. 1995, 45(9), 1594-1601) and has knownprotective properties against A-beta-induced toxicity in neurons. (a) C.Behl, J. B. Davis, R. Lesley, D. Schubert, Cell 1994, 77(6), 817-827; b)Z. Zhang, R. E. Rydel, G. J. Drzewiecki, K. Fuson, S. Wright, M.Wogulis, J. E. Audia, P. C. May, P. A. Hyslop, J. Neurochem. 1996,67(4), 1595-1606). Previous reports (Milton, N. G. N., et al.,Neuroreport 2001:12:2561-2566; Milton, N. G. N., Biochem. J.1999:344:293-296) indicate that the direct interaction between catalaseand some aggregated form of A-beta peptides can lead to the inactivationof catalase in vitro, suggesting that catalase-amyloid interactions maypotentially contribute to the cytotoxicity of A-beta in AD. The catalasewas assayed to determine whether it could associate with A-beta peptidesin fibrillar form and if small molecules that bind to A-beta fibrilscould be used to inhibit these catalase-A-beta fibril interactions.

FIG. 6 presents results from a standard ELISA assay (P. E. Fraser, L. K.Duffy, M. B. O'Malley, J. Nguyen, H. Inouye, D. A. Kirschner, J.Neurosci. Res. 1991, 28(4), 474-485) indicating that catalase indeedbinds to A-beta fibrils. After incubating A-beta fibrils (P. Inbar, J.Yang, Bioorg. Med. Chem. Lett. 2006, 16(4), 1076-1079) with a solutionof catalase, ThT was found to compete with catalase for binding toA-beta fibrils with an IC₅₀ for ThT of 170 μM (FIG. 6 b). There was amaximum total inhibition of ˜50% for the catalase-A-beta fibrilsinteraction at concentrations of ThT of 3 mM and higher. A significantlyhigher maximum total inhibition (compared to ThT) was observed of 91%for BTA-EG₄ and 92% for BTA-EG₆ for the interaction of catalase withA-beta fibrils (FIGS. 6 c and 6 d). An IC₅₀ of 590 μM for BTA-EG₄ and 36μM for BTA-EG₆ were found for the inhibition of binding of catalase toA-beta fibrils. These results indicate that BTA-EG₄ and BTA-EG₆ can formmore complete protein-resistive surface coatings on A-beta fibrilscompared to ThT.

Example 7 Inhibition of A-Beta Fibril-ABAD Binding Interaction

To further test the generality of using small molecules to inhibitprotein—beta fibril interactions, ThT and its derivatives were testedfor their ability to inhibit the interaction between A-beta fibrils andthe mitochondrial protein A-beta-binding alcohol dehydrogenase (ABAD).The interaction between ABAD and aggregated A-beta peptides has beenshown to lead to oxidative stress in cells and, therefore, may play apotential pathogenic role in AD. (Lustbader, J. W., et al. Science2004:304:448-452; J. Frackowiak, B. Mazur-Kolecka, W. Kaczmarski, D.Dickson, Brain Res. 2001, 907(1-2), 44-53). Inhibition of theinteraction between ABAD and aggregated A-beta using a decoy peptideappears to protect cells from oxidative stress, (Lustbader, J. W., etal. Science 2004:304:448-452) suggesting the development of methods toinhibit ABAD-amyloid interactions may be a target for therapy againstAD.

A standard ELISA assay was developed to measure ABAD binding to A-betafibrils (FIG. 7 a). ThT, BTA-EG₄, and BTA-EG₆ were then examined fortheir ability to inhibit the ABAD-A-beta fibril interactions. FIGS. 7b-7 d demonstrate that ThT and the BTA derivatives are all able to atleast partially inhibit the ABAD-A-beta fibril interactions (IC₅₀'s inthe range of 100-500 μM). Both BTA-EG₄ and BTA-EG₆ were more effectiveat competitive replacement of ABAD on the A-beta fibrils compared toThT. A ˜75% total inhibition of the protein-amyloid interactions atconcentrations of BTA derivatives above 4 mM compared to ˜56% totalinhibition of the ABAD-A-beta fibril interaction with ThT at similarconcentrations was observed.

An interesting observation was that BTA-EG₄ and BTA-EG₆ inhibitedcatalase-A-beta fibril interactions more effectively than ABAD-A-betafibril interactions. This observation may be due to the fact thatcatalase has been reported to interact with residues 25-35 of A-betapeptides, (Milton, N. G. N., et al., Neuroreport 2001: 12:2561-2566;Milton, N. G. N., Biochem. J. 1999: 344:293-296) which are within the-beta-sheet region of A-beta fibrils. (L. Li, T. A. Darden, L.Bartolotti, D. Kominos, L. G. Pedersen, Biophys. J. 1999, 76(6),2871-2878). Since ThT and BTA derivatives are believed to associate withthe -beta-sheet region of amyloid fibrils, (Lockhart, A., et al., J.Biol. Chem. 2005:280:7677-7684) it seems reasonable that these smallmolecules could effectively compete with proteins that bind in the sameregion along the fibrillar axis. ABAD, however, has been reported tobind to a more solvent exposed portion of A-beta (i.e., residues 1-20)(Powell, A. J., et al., J. Mol. Biol. 2000:303:311-327) than catalaseand may not be as susceptible to ThT and its derivatives for competitionfor binding to A-beta fibrils. A significant difference was not observedbetween BTA-EG₄ and BTA-EG₆ for inhibition of the catalase-amyloid orABAD-amyloid binding interactions. This observation suggests the BTAcomponent of the small molecules plays the more structurally significantrole compared to the small differences in the size of the oligoethyleneglycol chain for inhibiting the binding of these natural proteins toA-beta fibrils.

When the inhibition of the catalase-A-beta fibril interactions with theinhibition of the IgG-A-beta fibril interactions was compared using thetwo BTA derivatives, however, comparable results were observed in termsof maximal inhibition. This result was somewhat surprising in that themonoclonal IgG (clone 6E10) was raised against residues 3-8 of A-betapeptides, which is a solvent exposed region of A-beta. G. Y. Wen, S. Y.Yang, W. Kaczmarski, X. Y. He, K. S. Pappas, Brain Res. 2002, 954(1),115-122) Since an experimentally significant difference for inhibitionof the IgG-A-beta fibril interactions with BTA-EG₄ compared to BTA-EG₆was observed (FIG. 5), it is possible that the size of the oligoethyleneglycol chain attached to the BTA may play a role in the inhibition ofthis monoclonal IgG with the A-beta fibrils. Thus depending on theparticular A-beta-binding protein, both the BTA and oligoethylene glycolcomponents of the small molecules may play a significant role forinhibiting protein-amyloid interactions. The general observation thatthe BTA compounds showed significantly improved efficacy for inhibitingprotein-amyloid interactions, in terms of total inhibition compared toThT, might be due to potential differences in binding sites for BTA andThT along the fibril axis. (Powell, A. J., et al., J. Mol. Biol.2000:303:311-327)

Example 8 Synthesis of BTA Compounds

I-Iodo-3,6,9,12,15-pentaoxaheptadecanol and 1-iodo-3,6,9-trioxaundecanolwere prepared according to literature. (H. Bauer, F. Stier, C. Petry, A.Knorr, C. Stadler, H. A. Staab, Eur. J. Org. Chem. 2001, (17),3255-3278.)

A representative procedure for the synthesis of the oligoethylene glycolderivatives of BTA is as follows:1-Iodo-3,6,9,12,15-pentaoxaheptadecanol (0.18 g, 0.45 mmol) was coupledto 2-(p-aminophenyl)-6-methyl-benzothiazole (0.09 g, 038 mmol) withpotassium carbonate (0.39 g, 2.8 mmol) in dry acetone (4 mL) underreflux conditions. The acetone was removed and the residue was taken upinto dichloromethane, and separated from an insoluble precipitate(presumably excess potassium carbonate and potassium iodide). After theprecipitate was removed by filtration, the solution was washed withbrine, dried over sodium sulfate, and the solvent was removed underreduced pressure. The residue was purified via flash chromatographyusing 4% methanol in ethylacetate as the eluent to yield a yellow oil(isolated yield was 28%).

BTA-EG₄: ¹H-NMR (400 MHz, CD₃OD): δ=2.44 (s, 3H), δ=3.34 (t, 2H, 2.7Hz), δ=3.55 (t, 2H, 2.7 Hz), δ=3.60-3.69 (m, 10H), δ=3.68 (t, 2H, 2.7Hz), δ=6.70 (d, 2H, 8.6 Hz), δ=7.26 (dd, 1H, 8.4 Hz, 1.2 Hz), δ=7.65 (d,1H, 1.2 Hz), δ=7.74 (d, 1H, 8.4 Hz), δ=7.78 (d, 2H, 8.4 Hz); ¹³C-NMR(400 MHz, CD₃OD): δ=21.43, 43.93, 62.21, 70.91, 7135, 7137, 71.60,71.62, 73.66, 113.34, 122.09, 122.36, 128.85, 129.86, 135.37, 136.04,153.08, 153.11, 170.47; MS (ESI-positive): m/z 417.19 [MH⁺] (calc. mass416.18); FT-IR (on KBr in cm⁻¹): 816, 1095, 1181, 1340, 1453, 1483,1604, 2858, 2913, 3394.

BTA-EG₆: ¹H-NMR (400 MHz, CD₃OD): δ=2.47 (s, 3H), δ=3.71-3.53 (m, 24H),δ=6.74 (d, 2H, 8.4 Hz), δ=7.29 (d, 21-1, 8.4 Hz), δ=7.91-7.70 (m, 3H);¹³C-NMR (400 MHz, CD₃OD): δ=21.46, 43.95, 62.16, 70.55, 7130, 71.39,71.45, 71.46, 71.47, 71.51, 71.53, 71.56, 73.56, 113.38, 122.24, 122.38,128.86, 129.89, 135.24, 136.04, 152.93, 153.08, 170.33; MS(ESI-positive): m/z 505.25 [MH⁺] and 527.20 [MNa⁺] (calc. mass 504.23);FT-IR (on KBr in cm⁻¹): 820, 1097, 1184, 1260, 1345, 1455, 1481, 1610,2868, 2919, 3356.

Synthesis of BTA-EG₆-BTA

1,17-diiodo-3,6,9,12,15-pentaoxaheptadecane and1,14-diodo-3,6,9,12-tetraoxatetradecane were prepared according toliterature. (H. Bauer, F. Stier, C. Petry, A. Knorr, C. Stadler, H. A.Staab, Eur. J. Org. Chem. 2001, (17), 3255-3278)

1,17-diiodo-3,6,9,12,15-pentaoxaheptadecane was coupled to2-(p-aminophenyl)-6-methyl-benzothiazole with potassium carbonate in dryacetone under reflux conditions. The acetone was removed and the residuewas taken up into dichloromethane, and separated from an insolubleprecipitate (presumably excess potassium carbonate and potassiumiodide). After the precipitate was removed by filtration, the solutionwas washed with brine, dried over sodium sulfate, and the solvent wasremoved under reduced pressure. The residue was purified via flashchromatography using 4% methanol in ethylacetate as the eluent to yielda yellow oil.

BTA-EG₆-BTA: ¹H-NMR (400 MHz, CD₃OD): δ=2.43 (s, 6H), δ=3.21-3.29 (m,12H), &=3.71-3.53 (m, 8H), δ=4.01-4.07 (m, 4H), δ=6.67 (d, 2H, 8.4 Hz),δ=7.27 (d, 2H, 8.4 Hz), δ=7.91-7.70 (m, 3H); MS (ESI-positive): m/z727.24 [MH⁺] and 749.18 [MNa⁺] (calc. mass 726.4).

1,17-Iodo-3,6,9,12-tetraoxatetradecane (1.0 g, 2.2 mmol) was coupled to2-(p-aminophenyl)-6-methyl-benzothiazole (3.7 g, 2.2 mmol) withpotassium carbonate (4.8 g, 34.9 mmol) in dry THF under refluxconditions. The THF was removed and the residue was taken up intodichloromethane, and separated from an insoluble precipitate (presumablyexcess potassium carbonate and potassium iodide). After the precipitatewas removed by filtration, the solution was washed twice with water,dried over sodium sulfate, and the solvent was removed under reducedpressure. The residue was purified via flash chromatography using amixture of dichloromethane/ethylacetate (7:3) as the eluent to yield ayellow oil (yield 24%).

BTA-EG₅-I: ¹H-NMR (500 MHz, CD₃OD): δ=2.42 (s, 3H), δ=3.20 (t, 2H, J=6.8Hz), δ=3.58-3.65 (m, 14H), δ=3.67 (t, 2H, J=6.8 Hz), δ=3.69 (t, 2H,δ=6.8 Hz), δ=6.62 (d, 2H, 8.5 Hz), δ=7.20 (d, 2H, 8.5 Hz), δ=7.58 (s,1H,), δ=7.81 (d, 2H, 8.0 Hz), δ=7.84 (d, 21-1, 9.0 Hz), MS(ESI-positive): m/z 571.04 [MH⁺] and 593.00 [MNa⁺] (calc. mass 570.23).

BTA-Eg₅-1 (50 mg, 0.09 mmol) and lithium hydroxide (4.2 mg, 0.17 mmol)were dissolved in 10 mL of THF and refluxed for 20 h. The solution wascooled to room temperature and the solvent was removed under reducedpressure. The residue was taken up in a mixture ofdichloromethane/ethylacetate (50:50) and filtered through silica gel toremove excess lithium hydroxide. The crude material was purified viasilica gel flash chromatography using a mixture ofdichloromethane/ethylacetate (70:30) as the eluent to yield 22 mg of ayellowish oil (yield 56%). BTA-Azocrown-5: ¹H-NMR (400 MHz, CD₃OD):δ=2.47 (s, 3H), δ=3.59-3.70 (m, 16H), δ=3.79 (t, 4H, 6.0 Hz), δ=6.72 (d,2H, 8.8 Hz), δ=7.26 (s, 2H), δ=7.63 (s, 1H), δ=7.85 (d, 1H, J=8.4 Hz),δ=7.90 (d, 2H, J=8.8 Hz); MS (ESI-positive): m/z 443.23 [MH⁺] and 465.17[MNa⁺] (talc. mass 442.19);

Inhibition graphs of BTA-EG₆-BTA and BTA-Azo-crown-5 are presented inFIG. 8.

Example 9 Inhibition Assays Procedure for Inhibition Assays:

All incubation steps were done at 25° C. unless stated otherwise.Phosphate buffered saline (PBS, 10 mM sodium phosphate, 138 mM sodiumchloride, 2.7 mM potassium chloride, pH 7.4) and potassium phosphatebuffer (KPi, 200 mM potassium phosphate, 10 mM mercaptoethanol, pH 6.6)were prepared fresh for each experiment.

Growth of A-beta fibrils: Aβ fibrils were grown from synthetic Aβ (1-42)peptides by incubating the peptides (74 μM) in ultrapure water at 37° C.for 72 hours. Fibrils were characterized by electron and scanning probemicroscopy. (P. Inbar, J. Yang, Bioorg. Med. Chem. Lett. 2006, 16(4),1076-1079).

Recombinant expression of ABAD: ABAD was obtained from an externalcontract laboratory (e.g., Commonwealth Biotechnologies, Inc., Richmond,Va.) that cloned the DNA for ABAD (genebank number AF035555), andexpressed and purified the protein from a 1 L culture of E. coli using aliterature protocol. (Yan., S. D., et al., J. Biol. Chem.1999:274:2145-2156); S. D. Yan, Y. Shi, A. Zhu, J. Fu, H. Zhu, Y. Zhu,L. Gibson, E. Stem, K. Collison, F. Al-Mohanna, S. Ogawa, A. Roher, S.G. Clarke, D. M. Stern, J. Biol. Chem. 1999, 274(4), 2145-2156; S. D.Yan, et al., Biochim. Biophys. Acta 2000:1502:145-157). N-terminalsequencing using 15 cycles of Edman degradation was consistent with thepredicted sequence of the protein.

Qualitative determination of the binding of Aβ fibrils to catalase andARAD: The wells of a 96 well plate were coated with catalase or ABAD byincubating each well for 2 hours with 50 μL of a solution of catalase(24 nM in 1% BSA/PBS buffer) or ABAD (1.0 μM, in 1% BSA/KPi buffer).After removal of the solutions containing excess catalase or ABAD, allwells were blocked for 60 min. using 300 μL of a solution containing 1%BSA in PBS buffer to suppress non-specific adsorption of IgGs to thewells. Wells were washed with 300 μL of PBS buffer and incubated for 2hours with 50 μL of solutions containing Aβ fibrils (variousconcentrations were obtained by diluting a stock solution of 49 μMA-beta fibrils) in 1% BSA/PBS buffer. Wells were washed twice with 300μL of PBS buffer and each well was incubated for an hour with 50 μL of asolution containing a mouse monoclonal anti-Aβ IgG (clone 6E10, lot#145271, 1.1 nM in 1% BSA/PBS).

The amount of bound monoclonal IgGs was quantified by removing theexcess solution, washing the wells twice with 300 μL of PBS buffer andby incubating for 45 minutes with 50 μL of a polyclonal secondary rabbitIgG (anti-mouse IgG, 6.8 nM in 1% BSA/PBS) conjugated with alkalinephosphatase, followed by two washes with 300 μL of PBS buffer. Therelative amount of secondary IgG bound in each well was quantified byadding 50 μL of a solution containing p-nitrophenyl phosphate (NPP, 2.7mM, in 0.1 M diethanol amine/0.5 mM magnesium chloride, pH 9.8) to eachwell. The enzymatic hydrolysis reaction of NPP by alkaline phosphatasewas quenched after 45 min. by adding 50 μL of 0.25 N sodium hydroxidesolution to each well and quantifying the concentration ofp-nitrophenoxide at 405 nm using a UV-Vis microplate reader. Each datapoint from this assay represents the average of five independentmeasurements. Error bars represent standard deviations. Graphs werenormalized, plotted and fitted with the sigmoidal curve fitting optionin Origin 6.0 (Microcal Software, Inc., Northhampton, Mass., USA).

Inhibition of antiAβ IgG-A-beta fibril interactions using smallmolecules: The wells of a 96 well plate were coated with fibrils formedfrom A-beta peptides by incubating each well for 2 hours with 50 μL of a1.3 μM solution of Aβ fibrils in PBS. After removal of solutionscontaining excess A-beta fibrils, all wells were blocked for 60 min.using 300 μL of a solution containing 1% BSA in PBS buffer.

The BSA/PBS solutions were discarded and the wells were washed with 300μL of PBS buffer and incubated with 50 μL of an anti-An IgG (clone 6E10,Lot #145271, 1.1 nM in 1% BSA/PBS) for 1 hour. After removal ofsolutions containing excess IgG, 50 μL solutions of small molecules in1% BSA/PBS buffer (for ThT and BTA-EG₆) or 5% DMSO/1% BSA/PBS (forBTA-EG₄) (various concentrations were obtained by diluting a stocksolution) were incubated in the wells for 12 hours, followed by removalof solutions containing excess small molecule. The amount of monoclonalIgG present in the wells was quantified as described in the procedurefor determining the binding of Aβ fibrils to catalase and ABAD.

Inhibition of catalase-A-beta fibril or ABAD-A-beta fibril interactionsusing small molecules: The wells of a 96 well plate were coated withfibrils formed from A-beta peptides by incubating each well for 2 hourswith 50 μL of a 1.3 μM solution of Aβ fibrils in PBS. After removal ofsolutions containing excess A-beta fibrils, all wells were blocked for60 min. using 300 μL of a solution containing 1% BSA in PBS buffer.

The BSA/PBS solutions were discarded and the wells were washed with 300μL of PBS buffer and incubated with 50 μL of a human catalase solution(0.20 μM, in 1% BSA/PBS buffer) or 50 μL of an ABAD solution (10 in 1%BSA/KPi) at 37° C. for 3 hours or at 25° C. for 2 hours respectively.After removal of solutions containing excess catalase or ABAD, 50solutions of small molecules in 1% BSA/PBS buffer (for ThT and BTA-EG₆)or 5% DMSO/1% BSA/PBS (for BTA-EG₄) (various concentrations wereobtained by diluting a stock solution) were incubated in the wells for12 hours, followed by removal of solutions containing excess smallmolecule.

The wells were then washed twice with 300 μL of a solution containing 1%BSA in PBS and each well was incubated for 1 hour with 50 μL of asolution of a monoclonal mouse anti-catalase IgG (clone 1A1, lot #93195,2.2 nM in 1% BSA/PBS) or 50 μL of a solution of a monoclonal mouseanti-ABAD IgG (clone 5F3, lot #103614, 1.3 nM in 1% BSA/PBS buffer). Theamount of monoclonal IgG present in the wells was quantified asdescribed in the procedure for determining the binding of Aβ fibrils tocatalase and ABAD.

Example 10 Diffusion across Cell Membranes and the Blood Brain Barrier

In order to assess the potential utility of BTA-EG₄ and BTA-EG₆ asprobes to study protein-amyloid interactions in cellular assays, thelikeliness of the BTA derivatives to passively diffuse across cellmembranes and the Blood-Brain-Barrier (BBB) was estimated. (C. A.Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug DeliveryRev. 1997, 23(1-3), 3-25) Table 1 lists some of the intrinsic propertiesof ThT, BTA-EG₄, and BTA-EG₆ compared to some well-known parameters forthe biocompatibility of small molecules. (W. P. Walters, A. A. Murcko,M. A. Murcko, Curr. Opin. Chem. Bio. 1999, 3(4), 384-387) The logP_(octanol/water) was determined for the molecule.

15 μM solutions of small molecule were prepared in 5 mL PBS buffer. 5 mLof octanol was added to each aqueous solution of small molecule and thebiphasic layers were mixed by rapid vortexing. The mixture was thencentrifuged at 250×g to facilitate the formation of two clear layers.The layers were separated and the absolute concentrations of smallmolecules in each layer were quantified by measuring the absorbance ofthe layers at 348 nm and 412 nm for the BTA derivatives and ThT,respectively. The molar extinction coefficient of ThT and the BTAderivatives in octanol and PBS buffer were determined by comparison tostandard calibration curves of known quantities of small moleculesdissolved in octanol and PBS buffer. The partition coefficient wasexpressed as logarithm of the ratio of the concentration in octanoldivided by the concentration in PBS (i.e., log P).

Topological polar surface areas were estimated using MolinspirationCheminformatics software. This web-based software is available on theWorldWideWeb at molinspiration.com/cgi-bin/properties.

From the measured log P_(octanol/water) (Klunk, W. E., et al., Life Sci.2001, 69:1471-1484) and calculated polar surface areas (a) D. E. Clark,J. Pharm. Sci. 1999, 88(8), 815-821; b) J. Kelder, P. D. J. Grootenhuis,D. M. Bayada, L. P. C. Delbressine, J.-P. Ploemen, Pharm. Res. 1999,16(10), 1514-1519; P. Ertl, B. Rohde, P. Selzer, J. Med. Chem. 2000,43(20), 3714-3717) shown in Table 1, ThT may be predicted to haverelatively poor biocompatibility for use in cellular or in in vivostudies (previous reports agree with this prediction (Mathis, C. A., etal., Curr. Pharm. Design 2004, 10:1469-1492). The two oligoethyleneglycol derivatives of BTA, however, may be more suitable for cellularand in vivo studies than ThT since they fall within the measured andcalculated parameters for permeability to cells and the BBB. The uptakeof these molecules in the brain has yet to be determined.

TABLE 2 Chemical properties of ThT, BTA-EG₄, and BTA-EG₆ to assess theirlikeliness for passive diffusion through cell membranes and through theBlood-Brain-Barrier. (Li, L., et al., Biophys. J. 1999, 76: 2871-78;Lipinski, C. A., et al., Adv. Drug Delivery Rev. 1997, 23: 3-25). # of #of Molecular H-bond H-bond Compound weight Log (P)^(a) acceptors donorsTPSA^(b) Desired <500 g/mol  1-3 <10 <5 <120 Å² properties^(c) (ideal~2) BTA-EG₄ 418 g/mol 1.05 6 2 91.3 Å² BTA-EG₆ 504 g/mol 1.43 8 2 72.8Å² ThT 319 g/mol 0.43 1 0 7.1 Å² ^(a)determined by octanol-waterpartitioning ^(b)topological polar surface area (Clark, D. E., et al.,J. Pharm. Sci. 1999, 88: 815-821; Kelder, J., et al., Pharm. Res. 1999,16: 1514-1519), calculated with Molinspiration Cheminformatics software^(c)optimal properties for good permeability across lipophilicbiological barriers

In other examples of blood brain barrier penetration studies, rabbitsare injected with rifampin and the amount in the blood serum and thecelebrospinal fluid is determined after 2,3,6 and 12 hours. Samples aretaken under anesthesia as in, for example, Chan, K., et al., AsiaPacific J. Pharm. 1986, 1(1), 41-45.

Example 11 General Procedure for Detecting the Binding of SmallMolecules to Amyloid Fibrils

All incubation steps are done at 25° C. unless stated otherwise.Phosphate buffered saline (PBS, 10 mM sodium phosphate, 138 mM sodiumchloride, 2.7 mM potassium chloride, pH 7.4) are prepared fresh for eachexperiment.

Wells of a 96 well plate are coated with fibrils formed from variousamyloid forming peptides (such as α-synuclein, huntingtin, amylin . . .) by incubating each well for 2 hours with 50 μL of a solution ofamyloid fibrils in PBS (concentration 0.05-5 μM). After removal ofsolutions containing excess fibrils, all wells are blocked for 30 min.using 300 μL of a solution containing 1% BSA in PBS buffer.

The BSA/PBS solutions are discarded and the wells are washed with 300 μLof PBS buffer and 50 μL solutions of small molecules in 1% BSA/PBSbuffer (various concentrations can be obtained by diluting a stocksolution) are incubated in the wells for 12 hours, followed by removalof solutions containing excess small molecule. The wells are washedtwice with 300 μL, PBS buffer and incubated with 50 μL of a mousemonoclonal anti-amyloid IgG (IgGs are commercially available from e.g.,Abcam, Inc, Cambridge, Mass.) and are raised against the fibrildeposited into the wells. Concentrations are optimized and might rangefrom 0.05-10 nM in 1% BSA/PBS) for 1 hour. After removal of solutionscontaining excess IgG, the relative amount of secondary IgG bound ineach well is quantified by adding 50 μl, of a solution containingp-nitrophenyl phosphate (NPP, 2.7 mM, in 0.1 M diethanol amine/0.5 mMmagnesium chloride, pH 9.8) to each well. The enzymatic hydrolysisreaction of NPP by alkaline phosphatase is quenched after 45 min. byadding 50 μL of 0.25 N sodium hydroxide solution to each well andquantifying the concentration of p-nitrophenoxide at 405 nm using aUV-Vis microplate reader. Each data point from this assay will representthe average of five independent measurements. Error bars will representstandard deviations. Graphs can be normalized, plotted and fitted withthe sigmoidal curve fitting option in Origin 6.0 (Microcal Software,Inc., Northhampton, Mass., USA).

Specific Example for Detection of the Binding of Congo Red toAlpha-Synuclein:

The wells of a 96 well plate were coated with fibrils formed froma-synuclein peptides by incubating each well for 2 hours with 50 μL of asolution of a-synuclein amyloid fibrils in PBS (concentration 0.23 μM).After removal of solutions containing excess fibrils, all wells wereblocked for 30 min. using 300 μL of a solution containing 1% BSA in PBSbuffer.

The BSA/PBS solutions were discarded and the wells were washed with 300μL of PBS buffer and 50 μL solutions of congo red in 1% BSA/PBS buffer(various concentrations were obtained by diluting a stock solution) wereincubated in the wells for 12 hours, followed by removal of solutionscontaining excess small molecule. The wells were washed twice with 300μL PBS buffer and incubated with 50 μL of a mouse monoclonalanti-α-synuclein IgG (clone [4B12], Abcam, Inc., Cambridge, Mass., 0.7nM in 1% BSA/PBS) for 1 hour. After removal of solutions containingexcess IgG, the relative amount of secondary IgG bound in each well wasquantified by adding 50 μL of a solution containing p-nitrophenylphosphate (NPP, 2.7 mM, in 0.1 M diethanol amine/0.5 mM magnesiumchloride, pH 9.8) to each well. The enzymatic hydrolysis reaction of NPPwas quenched by alkaline phosphatase after 45 min. by adding 50 μL, of0.25 N sodium hydroxide solution to each well and quantifying theconcentration of p-nitrophenoxide at 405 nm using a UV-Vis microplatereader. Each data point from this assay represents the average of fiveindependent measurements. Error bars represent standard deviations.Graphs were normalized, plotted and fitted with the sigmoidal curvefitting option in Origin 6.0 (Microcal Software, Inc., Northhampton,Mass., USA).

An inhibition graph of alpha-synuclein fibrils with CongoRed is shown inFIG. 9.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Singular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “asubset” includes a plurality of such subsets, reference to “a nucleicacid” includes one or more nucleic acids and equivalents thereof knownto those skilled in the art, and so forth. The term “or” is not meant tobe exclusive to one or the terms it designates. For example, as it isused in a phrase of the structure “A or B” may denote A alone, B alone,or both A and B.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andsystems similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the methods, devices,and materials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of describing anddisclosing the processes, systems, and methodologies that are reportedin the publications which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Modifications may be made to the foregoing without departing from thebasic aspects of the invention. Although the invention has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, and yet these modifications and improvements are within thescope and spirit of the invention. The invention illustrativelydescribed herein suitably may be practiced in the absence of anyelement(s) not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. Thus, the terms and expressions which have been employed are usedas terms of description and not of limitation, equivalents of thefeatures shown and described, or portions thereof, are not excluded, andit is recognized that various modifications are possible within thescope of the invention. Embodiments of the invention are set forth inthe following claims.

1. A method of preventing or alleviating the symptoms of an amyloidassociated disease comprising contacting A-beta fibrils with a compoundof formula I:

wherein R₁-R₈ are selected from the group consisting of hydrogen,deuterium, tritium, fluoride, chloride, bromide, iodide, hydroxide,amino, methylamino, dimethylamino, trimethylammonium, methyl, ethyl,methoxy, ethoxy, fluoromethyl, difluoromethyl and trifluoromethyl,wherein at least one of R₅-R₈ and one of R₁-R₄ is H; and P is selectedfrom the group consisting of

wherein m is an integer between 1 and 20; n is 0, 1, or 2; q is aninteger between 1 and 20; R₉-R₁₆ are selected from the group consistingof hydrogen, deuterium, tritium, fluoride, chloride, bromide, iodide,hydroxide, amino, methylamino, dimethylamino, trimethylammonium, methyl,ethyl, methoxy, ethoxy, fluoromethyl, difluoromethyl andtrifluoromethyl, wherein at least one of R₉-R₁₂ and one of R₁₃-R₁₆ is H;and X is hydrogen, methyl, or ethyl.
 2. The method of claim 1, whereinsaid disease is a neuronal disease.
 3. The method of claim 2, whereinsaid neuronal disease is selected from the group consisting ofAlzheimer's disease, Parkinson's disease, Huntington's disease Down'sSyndrome, and spongiform encephalopathy.
 4. A method of preventing oralleviating the symptoms of an amyloid associated disease comprisingcontacting A-beta fibrils with a sufficient amount of a first bindingmolecule to decrease the interactions of said A-beta fibrils with asecond binding molecule.
 5. The method of claim 4, wherein said diseaseis a neuronal disease.
 6. The method of claim 4, wherein said secondbinding molecule is a cellular component in the brain.
 7. The method ofclaim 4, wherein said second binding molecule is selected from the groupconsisting of catalase, ABAD, and RAGE.
 8. The method of claim 4,wherein said first binding molecule is selected from the groupconsisting of Congo Red, a Congo Red derivative, Thioflavin T and aThioflavin T derivative.
 9. The method of claim 8, wherein said firstbinding molecule is a compound of formula I:

wherein R₁-R₈ are selected from the group consisting of hydrogen,deuterium, tritium, fluoride, chloride, bromide, iodide, hydroxide,amino, methylamino, dimethylamino, trimethylammonium, methyl, ethyl,methoxy, ethoxy, fluoromethyl, difluoromethyl and trifluoromethyl,wherein at least one of —R₅-R₈ and one of R₁-R₄ is H; and P is selectedfrom the group consisting of

wherein m is an integer between 1 and 20; n is 0, 1, or 2; q is aninteger between 1 and 20; R₉-R₁₆ are selected from the group consistingof hydrogen, deuterium, tritium, fluoride, chloride, bromide, iodide,hydroxide, amino, methylamino, dimethylamino, trimethylammonium, methyl,ethyl, methoxy, ethoxy, fluoromethyl, difluoromethyl andtrifluoromethyl, wherein at least one of R₉-R₁₂ and one of R₁₃-R₁₆ is H;and X is hydrogen, methyl, or ethyl.
 10. The method of claim 4,comprising administering a therapeutically effective amount of saidfirst binding molecule to an individual.
 11. A method for diagnosing anamyloid associated disease in an individual, comprising administering anA-beta fibril-binding compound to an individual and detecting thebinding of said compound to amyloid deposits in said individual, whereinsaid compound is selected from the group consisting of Congo Red,Fluoroscein, Acridine Orange, Diamino Acridine, Crystal Violet,Thioflavin T, Oxytetracycline, Tetracycline, Chlortetracycline, TannicAcid, Rosmarinic Acid, (+)-Catechin, (−)-Nicotine, Morin, Serotonin,Dopamine, Curcumin, (R)-Ibuprofen, (S)-Naproxen, Rifampin,D-(+)-Trehalose, D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and a compound of formula I:

wherein R₁-R₈ are selected from the group consisting of hydrogen,deuterium, tritium, fluoride, chloride, bromide, iodide, hydroxide,amino, methylamino, dimethylamino, trimethylammonium, methyl, ethyl,methoxy, ethoxy, fluoromethyl, difluoromethyl and trifluoromethyl,wherein at least one of R₅R₈ and one of R₁-R₄ is H; and P is selectedfrom the group consisting of

wherein m is an integer between 1 and 20; n is 0, 1, or 2; q is aninteger between 1 and 20; R₉-R₁₆ are selected from the group consistingof hydrogen, deuterium, tritium, fluoride, chloride, bromide, iodide,hydroxide, amino, methylamino, dimethylamino, trimethylammonium, methyl,ethyl, methoxy, ethoxy, fluoromethyl, difluoromethyl andtrifluoromethyl, wherein at least one of R₉-R₁₂ and one of R₁₃-R₁₆ is H;and X is hydrogen, methyl, or ethyl, and derivatives and analogsthereof.
 12. A method of preventing or alleviating the symptoms of anamyloid associated disease comprising contacting A-beta fibrils with asufficient amount of a pharmaceutical composition comprising apharmaceutically acceptable carrier and an A-beta fibril bindingcompound selected from the group consisting of Congo Red, Fluoroscein,Acridine Orange, Dimino Acridine, Crystal Violet, Thioflavin T,Oxytetracycline, Tetracycline, Chlortetracycline, Tannic Acid,Rosmarinic Acid, (+)-Catechin, (−)-Nicotine, Morin, Serotonin, Dopamine,Curcumin, (R)-Ibuprofen, (S)-Naproxen, Rifampin, D-(+)-Trehalose,D-nicotine, pegylated derivatives of1-(p-aminophenyl)-6-methylbenzothiazole, and a compound of formula I:

wherein R₁-R₈ are selected from the group consisting of hydrogen,deuterium, tritium, fluoride, chloride, bromide, iodide, hydroxide,amino, methylamino, dimethylamino, trimethylammonium, methyl, ethyl,methoxy, ethoxy, fluoromethyl, difluoromethyl and trifluoromethyl,wherein at least one of R₅-R₈ and one of R₁-R₄ is H; and P is selectedfrom the group consisting of

wherein m is an integer between 1 and 20; n is 0, 1, or 2; q is aninteger between 1 and 20; R₉-R₁₆ are selected from the group consistingof hydrogen, deuterium, tritium, fluoride, chloride, bromide, iodide,hydroxide, amino, methylamino, dimethylamino, trimethylammonium, methyl,ethyl, methoxy, ethoxy, fluoromethyl, difluoromethyl andtrifluoromethyl, wherein at least one of R₉-R₁₂ and one of R₁₃-R₁₆ is H;and X is hydrogen, methyl, or ethyl; and derivatives and analogsthereof, to decrease the interactions of said A-beta fibrils with asecond binding molecule.
 13. The method of claim 12, wherein saiddisease is a neuronal disease.